Imaginal Discs: The Genetic and Cellular Logic of Pattern Formation (Developmental and Cell Biology Series)

IMAGINAL DISCS With the elucidation of the complete ﬂy genome, traditional ﬂy genetics is in more demand than ever. Gene...

Author: Lewis I. Held | Jr.

48 downloads 1153 Views 5MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!

Report copyright / DMCA form

DOWNLOAD PDF

IMAGINAL DISCS With the elucidation of the complete ﬂy genome, traditional ﬂy genetics is in more demand than ever. Genetics will allow us to explain the role of each of the 14,000 genes, many of which are involved in the development of imaginal discs. These hollow sacs of cells make adult structures during metamorphosis, and their study is crucial to comprehending how a larva becomes a fully functioning ﬂy. This book examines the genetic circuitry of the well-known “fruit ﬂy,” tackling questions of cell assemblage and pattern formation, of the hows and the whys behind the development of the ﬂy. The book ﬁrst establishes that ﬂy development relies primarily on intercellular signaling, and then discusses how this signaling occurs. After an initial examination of the proximity versus pedigree imperatives, the book delves into bristle pattern formation and disc development, with entire chapters devoted to the leg, wing, and eye. Extensive appendices include a glossary of protein domains, catalogs of well-studied genes, and an outline of signaling pathways. More than 30 wiring diagrams, among 67 detailed schematics, clarify the text. The text goes beyond the Internet databases insofar as it puts these myriad facts into both a conceptual framework and a historical context. Overall, the aim is to provide a comprehensive reference guide for students and researchers exploring this fascinating, but often bewildering, ﬁeld. Lewis I. Held, Jr., is Associate Professor in the Department of Biological Sciences at Texas Tech University.

Developmental and Cell Biology Series series editors Jonathan B. L. Bard, Department of Anatomy, Edinburgh University Peter W. Barlow, Long Ashton Research Station, University of Bristol David L. Kirk, Department of Biology, Washington University The aim of the series is to present relatively short critical accounts of areas of developmental and cell biology where sufﬁcient information has accumulated to allow a considered distillation of the subject. The ﬁne structure of cells, embryology, morphology, physiology, genetics, biochemistry and biophysics are subjects within the scope of the series. The books are intended to interest and instruct advanced undergraduates and graduate students and to make an important contribution to teaching cell and developmental biology. At the same time, they should be of value to biologists who, while not working directly in the area of a particular volume’s subject matter, wish to keep abreast of developments relevant to their particular interests. books in the series 18. C. J. Epstein The Consequences of Chromosome Imbalance: Principles, Mechanisms and Models 19. L. Sax´en Organogenesis of the Kidney 20. V. Raghavan Developmental Biology of the Fern Gametophytes 21. R. Maksymowych Analysis of Growth and Development in Xanthium 22. B. John Meiosis 23. J. Bard Morphogenesis: The Cellular and Molecular Processes of Developmental Anatomy 24. R. Wall This Side Up: Spatial Determination in the Early Development of Animals 25. T. Sachs Pattern Formation in Plant Tissues 26. J. M. W. Slack From Egg to Embryo: Regional Speciﬁcation in Early Development 27. A. I. Farbman Cell Biology of Olfaction 28. L. G. Harrison Kinetic Theory of Living Pattern 29. N. Satoh Developmental Biology of Ascidians 30. R. Holliday Understanding Ageing 31. P. Tsonis Limb Regeneration 32. R. Rappaport Cytokinesis in Animal Cells 33. D. L. Kirk Volvox: Molecular Genetic Origins of Multicellularity and Cellular Differentiation 34. R. L. Lyndon The Shoot Apical Meristem: Its Growth and Development 35. D. Moore Fungal Morphogenesis 36. N. Le Douarin & C. Kalcheim The Neural Crest, Second Edition 37. P. R. Gordon-Weeks Neuronal Growth Cones 38. R. Kessin Dictyostelium 39. L. I. Held, Jr. Imaginal Discs: The Genetic and Cellular Logic of Pattern Formation

0 521 25464 7 0 521 30152 1 0 521 33022 X 0 521 35327 0 0 521 35053 0 0 521 43612 5 0 521 36115 X 0 521 24865 5 0 521 40943 8 0 521 36438 8 0 521 30691 4 0 521 35221 5 0 521 47802 2 0 521 44149 8 0 521 40173 9 0 521 45207 4 0 521 40457 6 0 521 55295 8 0 521 62010 4 0 521 44491 8 0 521 58364 0 0 521 58445 0

Thomas Hunt Morgan (3rd from right) and his associates at Columbia University. This luncheon was held in the “Chart Room” on 2 January 1919, to celebrate the return of Alfred Henry Sturtevant (foreground with beer and cigar) from his brief stint as a soldier in World War I [72, 651, 1556, 2283]. Calvin Bridges (center) is feigning a chat with a museum mannequin (Homo erectus) dressed in Sturt’s uniform. Clockwise from this anthropoid “guest” are Hermann J. Muller, T. H. Morgan (“the Boss”), Frank E. Lutz, Otto L. Mohr, Alfred F. Huettner, A. H. Sturtevant, Franz Schrader, Ernest G. Anderson, Alexander Weinstein, S. C. Dellinger, and Calvin B. Bridges. Curt Stern (not shown) did not join the team until 1924 [3071]. This merry band of pioneers launched a great quest for the secrets of genetics, and they had a knack for solving mysteries that rivaled Sherlock Holmes [72, 650, 651, 2951, 4182, 4184]. Nevertheless, as the informality of this party indicates, these legendary heroes did not take themselves too seriously [72, 3903]. Indeed, their lightheartedness has suffused this ﬁeld ever since [4696] and is reﬂected in the whimsical names of many ﬂy genes [2561]. Most of the mutations they studied affect the adult’s anatomy by altering the development of the larva’s imaginal discs. Those discs are the subject of this book, one of whose aims is to celebrate the triumph of the quest. This picture is from Sturt’s photo album. It was provided courtesy of the Archives, California Institute of Technology.

IMAGINAL DISCS The Genetic and Cellular Logic of Pattern Formation

LEWIS I. HELD, JR. Texas Tech University

CAMBRIDGE UNIVERSITY PRESS Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, São Paulo Cambridge University Press The Edinburgh Building, Cambridge CB2 2RU, UK Published in the United States of America by Cambridge University Press, New York www.cambridge.org Information on this title: www.cambridge.org/9780521584456 © Lewis I. Held, Jr. 2002 This book is in copyright. Subject to statutory exception and to the provisions of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. First published 2002 This digitally printed first paperback version 2005 A catalogue record for this publication is available from the British Library Library of Congress Cataloguing in Publication data Held, Lewis I., 1951– Imaginal discs : the genetic and cellular logic of pattern formation / Lewis I. Held, Jr. p. cm. Includes bibliographical references and index. ISBN 0-521-58445-0 1. Drosophila melanogaster – Genetics. 2. Drosophila melanogaster – Embryology. 3. Drosophila melanogaster – Morphogenesis – Molecular aspects. 4. Cellular signal transduction. 5. Cell interaction. I. Title. QH470.D7 H45 2002 576.5 – dc21 2001043553 ISBN-13 978-0-521-58445-6 hardback ISBN-10 0-521-58445-0 hardback ISBN-13 978-0-521-01835-7 paperback ISBN-10 0-521-01835-8 paperback

Contents

Preface

CHAPTER ONE. CELL LINEAGE VS. INTERCELLULAR SIGNALING

Discs are not clones No part of a disc is a clone, except claws and tiny sense organs Cells belong to lineage “compartments”

CHAPTER TWO. THE BRISTLE

Numb segregates asymmetrically and dictates bristle cell fates Delta needs to activate Notch, but not as a signal per se Amnesic cells can use sequential gating to simulate a binary code Notch must go to the nucleus to function E(spl)-C genes are Su(H) targets but play no role in the SOP lineage The transcription factor Tramtrack implements some cell identities Hairless titrates Su(H) Several other genes help determine the 5 cell fates Pox neuro and Cut specify bristle type Bract cells are induced by bristle cells Macrochaetes and microchaetes differ in size but not in kind

CHAPTER THREE. BRISTLE PATTERNS

Surprisingly, different macrochaete sites use different signals Prepatterns may contain hidden “singularities” How Achaete and Scute control bristles was debated for decades In 1989, Achaete and Scute were found to mark “proneural clusters” In 1995, the old AS-C paradigm toppled and a new one emerged Proneural “spots” shrink to SOP “dots” The SOP uses a feedback loop to raise its Ac and Sc levels Two other bHLH genes (asense and daughterless) assist SOPs

page xi

1 1 4 4

5 5 9 10 12 15 18 20 23 27 28 29

31 33 36 36 43 44 45 48 48

vii

viii

CONTENTS

“Lateral” or “mutual” inhibition ensures one SOP per PNC Notch-pathway and proneural genes are functionally coupled Doses of Notch-pathway genes can bias the SOP decision Extra SOPs could be inhibited by contact or diffusion (or both) Scabrous may be the diffusible SOP inhibitor Inhibitory ﬁelds dictate the spacing intervals of microchaetes Microchaetes come from proneural stripes, not spots Hairy paints “antineural” stripes on the legs Leg bristles use extra ﬁne-tuning tricks Chemosensory leg bristles are patterned like notal macrochaetes Extramacrochaetae superimposes an uneven antineural “mask” Dose dependency implies that HLH proteins “compute” bristles Robustness of patterning may be due to a tolerant time window Atonal and Amos are proneural agents for other types of sensilla Other (upstream) pathways govern bristle patterning

CHAPTER FOUR. ORIGIN AND GROWTH OF DISCS

Segmentation genes set the stage for disc initiation Prepatterns and gradients clashed in trying to explain homeosis Homeotic genes implement regional identities Wing and haltere discs “grow out” from 2nd- and 3rd-leg discs Thoracic discs arise at Wingless/Engrailed boundaries Cell lineage within compartments is indeterminate The Polar Coordinate Model linked regeneration to development But regeneration has peculiarities that set it apart

CHAPTER FIVE. THE LEG DISC

The Molecular Epoch of disc research was launched in 1991 Bateson’s Rule (1894) governs symmetry planes in branched legs Meinhardt’s Boundary Model deftly explained Bateson’s Rule The Boundary and PC Models jousted in a “Paradigm War” Hh, Dpp, and Wg are the chief intercellular signaling molecules P-type cells use Hh to “talk” to A-type cells nearby Hh elicits expression of Dpp and Wg along the A/P boundary Dpp dorsalizes and Wg ventralizes, or do they? Dpp and Wg are mutually antagonistic Dpp and Wg jointly initiate distal outgrowth But Dpp seems more crucial than Wg as a growth factor The A/P boundary can migrate when its “jailors” are “asleep” Regeneration is due to a Hh spot in the peripodial membrane The Polar Coordinate Model died in 1999 How Hh, Dpp, and Wg move is not known, nor is their range Whether Dpp and Wg travel along curved paths is not known Hairy links global to local patterning Questions remain about the Hh-Dpp-Wg circuitry Distal-less is necessary and sufﬁcient for distalization

49 50 52 52 53 55 57 61 62 67 68 71 73 74 75

76 76 80 84 85 87 91 92 96

97 97 99 100 103 105 107 109 111 114 115 118 119 122 123 124 125 128 128 129

CONTENTS

Proximal and distal cells have different afﬁnities Dachshund is induced at the Homothorax/Distal-less interface Homothorax and Extradenticle govern the proximal disc region Fasciclin II is induced at the BarH1/Aristaless interface BarH1 and Bric a` brac affect P-D identity, joints, and folds Leg segmentation requires Notch signaling

CHAPTER SIX. THE WING DISC

The A-P axis is governed by Hh and Dpp but not by Wg Dpp turns on omb and spalt at different thresholds Dpp regulates omb and spalt similarly despite clues to the contrary Dpp does not regulate tkv in 3rd instar despite clues to the contrary A vs. P identities might explain how a straight A/P line emerges But the A/P line appears to straighten via a signaling mechanism Intercalation is due to a tendency of Dpp gradients to rise The variable height of Dpp gradients makes them appear seamless A Wg gradient speciﬁes cell fates along the wing’s D-V axis Perpendicular (Dpp × Wg) gradients suggest Cartesian coordinates But cells do not seem to record positional values per se Wg’s repression of Dfz2 is inconsequential Apterous’s role along the D-V axis resembles Engrailed’s A-P role Chip cooperates with Apterous, and “Dorsal wing” acts downstream Serrate and Delta prod Notch to evoke Wg at the D/V line Fringe prevents Notch from responding to Serrate The core D-V circuit plugs into a complex network The wing-notum duality is established by Wg and Vein But Vestigial and Scalloped dictate “wingness” per se Straightening of the D/V border requires Notch signaling and Ap Straightening of veins may rely on similar tricks Two cell types predominate in the wing blade: vein and intervein Veins come from proveins that look like proneural ﬁelds But the resemblance is only superﬁcial All veins use the EGFR pathway But interveins also use the EGFR pathway (at a later time) Veins 3 and 4 are positioned by the Hh pathway Veins 2 and 5 are positioned by the Dpp pathway The Dpp pathway later implements the vein state A cousin of Dpp (Gbb) fosters the A and P cross-veins Vein 1 uses a combination of Dpp and Wg signals Macrochaetes are sited by various “prepattern” inputs How bristle axons get wired into the CNS is not known

CHAPTER SEVEN. THE EYE DISC

Compound eyes have ∼750 facets, with 8 photoreceptors per facet Unlike the bristle, the ommatidium is not a clone The eye has D and V compartments (despite doubts to the contrary)

ix

132 132 133 134 135 135

137 137 140 143 143 148 149 153 155 156 157 158 158 158 160 161 164 165 167 171 173 173 174 175 175 177 184 185 186 188 188 189 190 191

197 197 201 202

x

CONTENTS

The Iroquois Complex controls D-V polarity via Fringe and Notch A morphogenetic wave creates the ommatidial lattice D-V polarity depends on a rivalry between R3 and R4 precursors R1–R8 cells arise sequentially, implying a cascade of inductions But the ﬁnal cell (R7) is induced by the ﬁrst one (R8) Various restraints prevent more than one cell from becoming R7 The information content of the inductive signals may be only 1 bit No transcription factor “code” has yet been found for R cells The lattice is created by inhibitory ﬁelds around R8 precursors The lattice is tightened when excess cells die Eye bristles arise independently of ommatidia The MF operates like a moving A/P boundary Dpp and Wg control the rate of MF progress The MF originates via different circuitry

CHAPTER EIGHT. HOMEOSIS

BX-C and ANT-C specify gross metameric identities along the body Ubx enables T3 discs to develop differently from T2 discs But Ubx does so by directly managing target genes in multiple echelons Pc-G and Trx-G “memory” proteins keep homeotic genes on or off Homothorax, Distal-less, and Spineless specify leg vs. antennal fates If a “master gene” exists for the eye, then it is also a micromanager The manifold “enhanceosome” is a wondrous Gordian Knot The deepest enigma is how evolution rewired the circuit elements

203 208 209 211 213 213 216 218 224 227 228 229 234 234

237 237 243 244 247 249 252 254 254

EPILOGUE

256

APPENDIX ONE. Glossary of Protein Domains

257

APPENDIX TWO. Inventory of Models, Mysteries, Devices, and Epiphanies

266

APPENDIX THREE. Genes That Can Alter Cell Fates Within the (5-Cell)

Mechanosensory Bristle Organ

271

APPENDIX FOUR. Genes That Can Transform One Type of Bristle Into

Another or Into a Different Type of Sense Organ

276

APPENDIX FIVE. Genes That Can Alter Bristle Number by Directly Affecting

SOP Equivalence Groups or Inhibitory Fields

278

APPENDIX SIX. Signal Transduction Pathways: Hedgehog,

Decapentaplegic, and Wingless

285

APPENDIX SEVEN. Commentaries on the Pithier Figures

297

References

307

Index

441

Preface

How embryos “self-assemble” has fascinated thinkers for millennia [2918, 3064, 3190]. Among the ancient Greeks, Aristotle (384–322 bce) made copious observations and coined the term “morphogenesis,” which is still in use today [2989, 4305]. For the past century, the science of “developmental mechanics” has hammered at this problem relentlessly, but it is only in the last decade that the core mysteries have ﬁnally cracked [1487]. The deepest secrets have come from a fairylike ﬂy named Drosophila melanogaster, probably the same species of “gnat” that Aristotle himself noticed hovering over vinegar slime [217, 3361, 4184]. Unfortunately, these insights can only be fully appreciated in the arcane language of ﬂy genetics. Hence this book full of runes and rules. This book concerns cuticular patterns, the cellular machinery that makes them, and the genetic circuitry that runs the machinery. Although it is mainly a survey, it is also a narrative that traces the roots of our knowledge. The story that it tells – albeit in condensed form – rivals the Iliad in scope (legions of researchers devoting decades to attacking thousands of genes) and the Odyssey in wonderment (monstrous mutants posing riddles that challenge even the most clever explorerheroes). Indeed, truth is often stranger than a fairy tale in the realm of the ﬂy. Believe it or not, there are even remote islands where giant drosophilids with dappled wings and feathery legs have been spied dancing and ﬁghting in the misty forests [668, 669]. Ever since 1910 when T. H. Morgan’s ﬁrst “ﬂy paper” was published [2948], the ﬁeld of ﬂy genetics has brimmed with intriguing curiosities [820, 2951, 3673] and equally colorful human personalities [120, 327, 2283, 4183]. Added to these delights is a menagerie of recently discovered molecules

– e.g., the midget “Bearded” (81 a.a.) [2499] and the giant “Dumpy” (3680 a.a.) [4668]. Now that the ﬂy genome project is ending [14], the world is peering into this circus. What newcomers may not realize is that this ﬁeld offers many diversions beyond its databases. Like other holometabolous insects, ﬂies live two lives – ﬁrst as a grub, then as a ﬂying adult [82]. During metamorphosis, 19 “imaginal discs” erupt from inside the maggot and are quilted together to form most of the adult skin. The gold-colored cuticle secreted by that skin is exquisitely ornate. The head is embossed with hundreds of domes that focus light onto bundles of photoreceptors, the thorax is sculpted into dozens of jointed parts that form a contraption for walking and ﬂying, and the abdominal wall (built from non-disc tissue [2648]) is pleated into an expansible chamber for digestion and reproduction. Nearly everywhere, the body surface sprouts bristles whose patterns can be as orderly as soldiers on parade. Why do only some cells make bristles? That is a problem of differentiation. Why do bristles arise only at certain sites? That is a problem of pattern formation, and these questions can be asked for structures in general. Beneath both problems is a coding enigma: how does the ﬂy’s 1-dimensional genome encode the 2dimensional cuticular landscape? Once, it seemed that each body part might be governed by its own set of genes [4509, 4512], but this notion proved wrong [1094, 1114, 2410, 4643]. In fact, most patterns are built by the same ensembles of genes. These modules arose eons ago in the mythical common ancestor of insects and vertebrates [1439, 3840]. Since then, evolution has customized the circuitry by making new intra- and inter-modular links [968, 1440].

xi

xii

What is the nature of the circuitry, and how does it program cells to “compute” patterns? That is the subject of this book. Topics are arranged roughly in order of increasing complexity. Chapter 1 establishes one simple fact: in contrast to nematodes, ﬂies rely primarily on intercellular signaling (vs. cell lineage) to assign cell fates. The rest of the book traces how signaling occurs. Chapter 2 delves into the 5-cell cluster that constructs a mechanosensory bristle. The bristle is an exception to the signaling rule: its cell fates are dictated almost entirely by lineage. Chapter 3 uses bristle patterns to show how cells communicate in populations larger than a bristle but smaller than a disc, and Chapter 4 sets the stage for a discussion of larger-scale patterning by reviewing how discs arise and grow. Chapters 5 to 7 explore how leg, wing, and eye discs use similar toolkits of genes in idiosyncratic ways. The other two major discs – haltere and genital – are excluded because their strategies so closely resemble wing [16, 51, 3875, 4683, 4684] and leg discs [679, 735, 1163, 2343, 2942, 3732], respectively. (Fly genitalia are evolutionarily modiﬁed appendages [1137, 1179, 1562].) Chapter 8 contemplates the phenomenon of homeosis in the context of evolution. Overall, the book’s quest is to understand cellular “epistemology” (what do cells know?) and “psychology” (how do they think?). Its approach involves de- and reconstruction: to cut through the jargon, tease out the facts, and then try to make sense of the models by piecing the clues back together using a priori reasoning. The bad news is that there are so many pieces in the puzzle that persistence will be needed. The good news is that their interactions are so limited that no fancy math is required to learn the rules of the game [3588, 3841]. A recurrent theme in the saga is how cellular riddles were solved by molecular genetics. The abiding moral is that there is much more experimental work to be done if we are to comprehend how the ﬂy’s ∼14,000 genes [14, 1559, 3618, 3674] – or a large portion thereof [280, 615, 963, 4273] – are orchestrated during patterning [698, 2162, 2237, 2845, 4084]. In short, the ﬂy still holds many secrets, and genomics will need genetics to ferret them out [465]. Thus, the book is a sampler of case studies and gedanken exercises, not an encyclopedia. That function is served by the Internet databases, and readers should consult two main websites: FlyBase ( ﬂybase.bio.indiana.edu) [124, 279] and The Interactive Fly (sdb.bio.purdue.edu) [484]. Fly lore is best savored by browsing the classics: the 1993 Cold Spring Harbor 2volume compendium on development [238], its gargan-

PREFACE

tuan 12-volume predecessor The Genetics and Biology of Drosophila [122], Mike Ashburner’s huge “handbook” [118], Lindsley and Zimm’s dictionary of ﬂy genes [2561], Bridges and Brehme’s Barnumesque catalog of freakish mutants [470], and the Morgan team’s magnum opus of 1925 [2951]. However, the fun of ﬂy research is best portrayed in the charming Fly by Martin Brookes (2001, Harper-Collins, N.Y.). Despite this disclaimer about breadth, a few topics are covered in depth in the appendices. Appendix 1 is a glossary of protein domains. Appendix 2 lists most of the ideas that have guided research in this ﬁeld. Appendices 3 to 5 catalog the well-studied genes that affect bristles, sensilla, or bristle patterns, and Appendix 6 outlines three of the key signaling pathways in disc development (Hedgehog, Wingless, and Decapentaplegic). The other two pathways are discussed in Chapters 2 (Notch) and 6 (EGFR). Appendix 7 contains additional comments about the ﬁgures. Historically, disc research has been reviewed intermittently. Disc histology was codiﬁed by Dietrich Bodenstein in 1950 [377]. Disc development and genetics were surveyed by Gehring and N¨othiger (1973) [1421], Postlethwait and Schneiderman (1973) [3448], Bryant (1978) [526], Shearn (1978) [3881], Poodry (1980) [3422], and Oberlander (1985) [3165]. The ﬁrst blush of moleculargenetic data was evaluated by Stephen Cohen in 1993 [834], and the fundamentals of signaling were summarized by Seth Blair in 1999 [358]. Two books that nicely bracket the last 30 years of investigation are The Biology of Imaginal Discs (1972, H. Ursprung and R. N¨othiger, eds.) [4426] and Developmental Genetics of Drosophila (1998, A. Ghysen, ed.) [1452]. Conventional nomenclature is used. Locations of genes are stated in terms of the salivary gland chromosome map [2561]: the 3-part code (e.g., “92E12–14”) denotes the chromosome section (1–20 span the X, 21–60 the 2nd, 61–100 the 3rd, and 101–102 the tiny 4th chromosome), the lettered subdivision (A–F), and the band or range of bands. Genes are italicized, but gene complexes (e.g., Bar-C) are not. All proteins are in plain type. Mutations are superscripted (e.g., numbLOF ), whereas wild-type alleles are not (numb) or are labeled with “+” (numb+ ). Null alleles are designated by a “null” or “−” superscript. Most gene names record the dominant (capital) or recessive (lowercase) nature of early mutations (e.g., Notch vs. numb). Capital “D” (Drosophila) is used for paralogs within the species (e.g., Dfz2 [310] in the frizzled series), whereas lowercase “d” refers to

PREFACE

orthologs of vertebrate genes (e.g., dTcf [692, 1517]). Proteins are always capitalized (e.g., Numb). Given these rules, the normal symbols for Hairless (H ) and hairy (h) are distinct for the genes but not for the proteins (“H” in both cases), so “H” will be used only for Hairless, while “Hairy” will be written out. Likewise, Beadex will be written out to avoid confusion with the protein encoded by bithorax (both would be “Bx”). Small capitals are employed for Boolean states (on, off), conditions (if, then, not), and conjunctions (and, or). Amino acid and nucleotide sequences are underlined. Boundaries are denoted by slash marks (e.g., “A/P”) and axes by hyphens (e.g., “A-P”). Short gene names (≤5 letters) are not usually abbreviated. Abbreviations include a.a. (amino acid), AEL (after egg laying), AP (after pupariation) a.k.a. (also known as), b.p. (base pair), h (hour), hs (heat shock), kb (kilobase), kD (kiloDalton), MC (macrochaete), mC (microchaete), St. (stage of embryogenesis), t.s. (temperaturesensitive), pers. comm. (personal communication), and unpub. obs. (unpublished observations). Times (h AEL or h AP) refer to a culture temperature of 25◦ C, unless stated otherwise. Polypeptide lengths are for the unprocessed (nascent) precursor. Genes that are usually called “neurogenic” (based on mutant phenotype) [436] are here termed “antineural” (based on function) [4387] to contrast them with “proneural” (based on function) genes [2018]. “Eye disc” refers to both the eye and antennal parts, and “wing disc” denotes the entire dorsal mesothoracic disc (wing, notal, and pleural parts). By tradition (quirky though it may be), fate maps employ left legs (Ch. 5), right wings (Ch. 6), and left eyes (Ch. 7) [185, 320, 526, 531], although right eyes are used by some authors [2962]. Readers must be familiar with the basics of ﬂy development [358, 2434, 3517] and the methods of modern genetics [354, 4671], including (1) induction of cell clones by ﬂp-mediated recombination [1530--1532, 3952, 4781] and the ﬂp-out trick [4159], (2) regional misexpression of genes via Gal4-UAS constructs [435, 3857], (3) temporal misexpression via heat-sensitive alleles [4214] or heat-shock promoters [2953], (4) enhancer trapping using lacZ reporter genes [278, 329, 1286, 4687], and (5) two-hybrid screening for protein interactions [222, 763, 1228, 1229, 1316]. Wherever possible, circuits are formulated in terms of Boolean logic [399] because this format shows syntax better than the “spaghetti diagrams” of genetics, electronics, or neural networks [2870]. The temptation to compare ﬂy circuits with vertebrate or nematode circuits

xiii

is generally resisted here for the sake of conciseness. Such comparisons can be found in Eric Davidson’s book Genomic Regulatory Systems [968] and at Tom Brody’s website The Interactive Fly. The term “link” is used in the sense of “causal linkage.” Links are symbolized as “ ” (activation) or “ ” (inhibition). When a gene is the object (e.g., “Dpp omb”), the effect is always at the transcriptional level, but pathways may be distilled in terms of either genes (en ci ptc) or proteins (En Ci Ptc), and any attendant ambiguities will be clariﬁed by context. Epistatic links need not be direct. Thus, “a c” could reﬂect a longer chain such as “a b c” or “a b c.” The reason for listing so many links in this book is to facilitate Aristotle’s goal of delineating the entire chain of causes from the fertilized egg to the adult [1993, 2919, 4305]. Only by concatenating all the known fragments can we see the gaps that remain to be ﬁlled. The terms “LOF” (Loss of Function) and “GOF” (Gain of Function) typically denote decreases or increases in levels of gene activity (i.e., under- or overexpression) [1117, 1455], but in the broader sense that will be used here, GOF also includes ectopic misexpression where the “gain” is regional (cf. Fig. 6.13). For example, clones of cells that express a wild-type allele of engrailed (en+ ) outside the territory where en+ is normally transcribed will be called “enGOF ” [4848]. Cases do arise where overexpressing a wild-type allele has effects that differ from expressing a constitutively active construct [3545], and these will be so indicated. Mutations that are neither LOF nor GOF (e.g., neomorphs and antimorphs) are rarer, and allele-speciﬁc superscripts will be retained for them (e.g., ci D [3818] and en1 [1636]). LOF and GOF tests are used to assess the necessity (LOF) and sufﬁciency (GOF) of a speciﬁc gene for a particular process [173, 3643, 4333, 4671], and they are valuable tools. However, neither is foolproof. For example, if we delete gene “a” and see no effect on bristles (a negative LOF result), then a is clearly dispensable for bristle formation, but we cannot conclude that a is irrelevant because it might be acting redundantly with gene “b” [2845, 4584]: “a or b bristle.” GOF data can also be misleading [6, 682, 1329]. For instance, if we drive the expression of gene “a” in a region where it is not normally transcribed and ﬁnd that it induces bristles (a positive GOF result), then a is clearly sufﬁcient for evoking bristles [1458, 1854, 2019, 3267], but this does not mean that a promotes bristle formation in wild-type ﬂies because GOF perturbations can saturate limiting components (e.g., bHLH

xiv

PREFACE

partners [438, 918, 1854] or external ligands [421]) or provoke interactions with other pathways (e.g., converging RTK cascades [326, 1117, 2623] or branched Frizzled chains [3912, 4365, 4867]), resulting in all sorts of artifacts [6]. Researchers beware!

the subtleties. Around every “gospel truth” there is a Talmudic aura of uncertainty. The author’s goal should be to make the material as accessible as possible without hiding any ambiguities. This book will attempt to do just that.

It is . . . unsafe to deduce normal gene function [when] the product is forced into inappropriate cells, perhaps in the absence of proteins with which it normally interacts and the presence of others that it does not normally encounter. [1304]

Nature is a home handywoman. Constrained by evolution, she does the job with the tools at hand, using a screwdriver for a hammer if necessary. . . . This machinery is neither elegant nor simple, but consists rather of a complex set of interacting proteins that were cobbled together by evolution. . . . Models help to organize our thoughts and offer testable hypotheses. Of course, in constructing a model, some data may need to be hammered into place, and the inconvenient data that cannot be coaxed into place have to be left out. The models that are frequently illustrated in minireviews . . . thus cannot be viewed as the ‘‘truth,” or they would narrow thought processes and squelch novel lines of research. We must be thoughtful iconoclasts, remembering that ultimately all models are wrong, fundamentally flawed or lacking the full complexity of systems shaped by evolution rather than intelligent design. We will thus use this forum to critique rather than prop up our model. It is increasingly clear that life is more complicated than portrayed there. [894]

Results derived from mutant analyses or from utilizing ectopic expression of a gene product reveal the potential of a particular interaction to occur, not whether the interaction actually occurs during wild-type development. [3248]

Artifacts can be minimized by combining LOF and GOF tests [147, 3462]. Indeed, that is the only way to distinguish factors that are “instructive” for cell fates from those that are merely “permissive.” Instructive agents have both LOF and GOF effects, whereas permissive agents have a LOF but no GOF effect [449, 1455]. Even this 2-pronged approach may not be able to resolve epistatic relations, however, where (1) interactions are cooperative as in multiprotein complexes, (2) pathways are nonlinear, (3) feedback obscures causality, or (4) the “upstream” vs. “downstream” ranking of genes contradicts the order of cellular actions in time. An example of the last difﬁculty involves scute and Notch. In general, scute is epistatic to Notch (i.e., scuteLOF NotchLOF ﬂies show the scuteLOF missing-bristle trait instead of the NotchLOF extra-bristle trait) [918, 1797, 1802, 3270, 3983], so scute should be acting downstream of Notch, but in fact scute must endow cells with “proneural competence” before Notch can enforce any “lateral inhibition” (cf. Ch. 3). The situation is even more complex at certain sites where Notch also acts before scute during a “prepattern” (pre-proneural) stage [461, 886]. Not all the ﬂy’s circuitry is as inscrutable as the Notchscute-Notch cascade, but our view of every subsystem is distorted by the imperfect lens of genetic dissection [2917, 3881, 4085, 4671]. Conclusions must therefore be qualiﬁed by layers of caveats about this or that alternative interpretation. The problem with such equivocation, of course, is that it can put readers to sleep. How much of this blather can readers tolerate? Why not just present “best guess” models and avoid all the dithering? Good advice on this issue comes from a delightful little essay entitled, “Wingless signaling: The inconvenient complexities of life.” Therein, Rachel Cox and Mark Peifer argue that cartoon-like abstractions are essential but must be tempered by critiques that convey

Only by venturing into the ocean of literature can novices experience the richer Fly World beyond the Internet harbors. Alas, it is all too easy to get lost in those rougher seas. For that reason, an effort is made to supply the equivalents of charts and buoys. To wit, all key mysteries that have taunted investigators are set in boldface when introduced. So are the models and metaphors that have been contrived to explain the mysteries, plus the epiphanies encountered whenever great mysteries were slain. All these concepts are inventoried in Appendix 2. Some of the coined names for the concepts are whimsical, but no more so than the silly names of many ﬂy genes. Indeed, working in this ﬁeld has been so much fun because of its playful irreverence – a legacy of the neophyte pioneers in Morgan’s team [119]. Even “the Boss” himself loved to clash ideas [2947] and smash idols [2946]. Ideas are contrasted here wherever possible, and the style is decidedly iconoclastic. All statements are source-referenced, and crossreferences that are not direct attributions are listed as “cf. such-and-such” – a style that is common in the humanities but rare in the sciences [1630]. The cf.’s mean to compare, confer, or just “see also.” Due to space limitations, some citation strings had to be truncated. Those cases are ﬂagged with a “” superscript to alert readers who want to trace earlier sources thereby. An unabridged bibliography is posted at The Interactive Fly.

PREFACE

Esoterica are banished to tables, ﬁgures, and appendices wherever possible, and supportive evidence is crammed into indented blocks of text so that readers can skip them if they want. Even so, readers may ﬁnd some sections of the text unnavigable without looking up the cited papers and tracing their lines of reasoning. Subheadings are worded as sentences so that the Table of Contents reads like a summary for each chapter. Gene abbreviations are deﬁned wherever they are used in the text. Overall, the layout is designed to avoid boring the expert without confusing the novice. I still remember how hard it was to make my way into this ﬁeld as an apprehensive apprentice. This ﬁeld has seen paradigm clashes of Promethean proportions, and those wars must be recounted to do the subject justice. For that reason, the modern facts have been woven into a historical tapestry, with a few homilies stitched in for good measure. Admitting past mistakes can help in spotting future pitfalls . . . even in the Olympian realm of molecular genetics, which surprisingly has more than a fair share of mortal foibles [1879, 2414, 3909, 4669, 4673]. The potential pitfalls include not only (1) the aforementioned LOF and GOF artifacts, but also (2) reporter anomalies (e.g., perdurance of β-gal [3764, 4188]), (3) antibody limitations (e.g., misleading epitopes on proteins that are cleaved [155, 3271] or reshaped [1980]), (4) confocal illusions [3293, 4760], and (5) in vitro inﬁdelities relative to in vivo conditions [655, 871]. For the next generation of researchers, some of the parables may sound quaint, but for those of us who toiled through this period, they are a chronicle worth preserving. Readers accustomed to color photos may bemoan the book’s reliance on black-and-white diagrams. I am sorry for any disappointment. The latter style just seemed more ﬁtting for an abstract analysis. All the ﬁgures were drawn in adobe Illustrator by me (a hopeless attempt to compete with my truly artistic siblings). They evolved from cartoons into montages. When many grew too big to ﬁt the standard 6 × 9-in. size of this series, I tried breaking them into pieces but found that the surgery was lethal. The montages had acquired a life of their own. They tell whole stories (some of which spill over into App. 7). I thank Cambridge for approving a

xv

larger trim size and for letting me set my own deadline. The cusp of the millennium seemed an apt time to step back and take a wide-angle “snapshot” of this blossoming ﬁeld. The last batch of citations came from the annual Drosophila Research Conference (in Washington, DC) entitled, “2001: A Fly Odyssey.” This project began in 1992 when Robin Smith (then Life Sciences Editor at Cambridge) asked me to write a book for this series at the behest of Paul Green (a series editor). The topic took shape gradually, and the contract was signed in 1996. By 1997, my other professional pursuits had to be sidelined as the writing became allconsuming. I thank Peter Barlow (another series editor) for calming my fears and Ellen Carlin (Assistant Life Sciences Editor) for trusting my judgment. Encouragement was provided by my dear parents (Maj. Gen. Lewis I. Held and Minnie Cansino Held), siblings (Lloyd, a.k.a Grey, and Linda), other relatives and sundry friends – most of whom remain skeptical that any sane adult can adore ﬂies. Maybe this book will change their minds? Probably not! Critical comments on portions of the manuscript were kindly furnished by Seth Blair, Tom Brody, Ian Duncan, Matt Gibson, Robert Holmgren, Teresa Orenic, Grace Panganiban, Amy Ralston, Allen Shearn, David Sutherland, and Tanya Wolff. The idea about Notch and Argos in the Skeptic-Theorist debate (Ch. 6) was Seth’s. I regret any overlooked errors. As one foot soldier in the global army of ﬂy pushers, I have met many “generals” over the years who ﬁgure prominently in this saga. By far the greatest – and humblest – was Curt Stern. His musings on the mysteries of patterning were the siren songs that lured me to this lovely ﬂy. Those of us who heeded his call have long dreamt of ﬁnding insights one day. Little did any of us suspect, though, that the bounty of revelations in the last decade would go so far beyond merely sating our curiosity. As we sift the treasure, the sparkle of so many answers is fostering – even in the saltiest among us – a profound sense of awe. Lewis I. Held, Jr. Lubbock, Texas April 2001

CHAPTER ONE

Cell Lineage vs. Intercellular Signaling

Imaginal discs are hollow sacs of cells that make adult structures during metamorphosis. They are so named because “imago” is the old term for an adult insect [4008], and their shape is discoid (i.e., ﬂat and round like a deﬂated balloon) [377]. They arise as pockets in the embryonic ectoderm and grow inside the body cavity until the larva becomes a pupa, at which point they turn inside out (“evaginate”) to form the body wall and appendages [3165]. In a D. melanogaster larva there are 19 discs (Fig. 1.1). Nine pairs form the head and thorax, and a medial disc forms the genitalia. The abdominal epidermis comes from separate cell clusters called “histoblast nests” [2301, 2648, 3647]. Unlike discs, histoblast nests remain superﬁcial during larval life [927] and do not grow until the pupal stage [2650]. Given the diversity of cell types in the adult skin (e.g., bristles, sensilla, photoreceptors) and the commonality of their descent from one progenitor (the fertilized egg), it is natural to ask how cells specialize to adopt divergent roles. In principle, cells can acquire instructions from ancestors or contemporaries [1654]. More speciﬁcally, a cell can inherit predispositions from its mother (“intrinsic” mode), take cues from neighbors (“extrinsic” mode), or both [477, 1614, 2019, 2451, 3741]. The predispositions could be gene states, while the cues could be diffusible ligands [1144, 3182]. To the extent that fates are assigned intrinsically, there should be a rigid correspondence between (1) parts of the anatomy and (2) branches of the lineage tree [1362, 4086, 4087]. That is, a clone of cells descended from ancestral cell “x” should make structure “X”, while another clone descended from ancestor cell “y” should make structure “Y”. Moreover, these rules should be obeyed

in every member of the species. C. elegans worms adhere closely to this strategy [1284, 4201, 4202], but ﬂies do not [1839, 1881]. In D. melanogaster, the only adult structures that use an intrinsic mode are tiny sense organs [532, 1410, 3441]. All larger parts of the body use extrinsic mechanisms. Thus, the problem of how discs develop can be reduced to questions about how cells communicate [695]. Who signals to whom? Over what distance? With what molecules? To what end?

Discs are not clones Proof that cell pedigrees are irrelevant for disc patterning was ﬁrst provided in a 1929 paper [4180] by Alfred Henry Sturtevant (1891–1970) – a wunderkind of the Morgan lab [257, 2504, 2615]. Sturtevant studied a strain that produced freakish ﬂies called “gynandromorphs” [2950]. Each such ﬂy is a patchwork of purely male and female tissues (Fig. 1.2) [1715]. They begin life with two X chromosomes but typically lose an X during the ﬁrst mitosis, so that one of the two zygotic nuclei becomes 1X [1695]. Because gender in ﬂies is dictated by the number of Xs relative to the numbers of autosomes [817], the 1X nucleus – and the half of the body that it populates – becomes male. Sexual traits are expressed autonomously at a single-cell level because ﬂies lack circulating sex hormones. The male/female boundary can be mapped throughout the cuticle (not just in dimorphic organs) by using recessive mutations to mark one of the Xs. The yellowLOF mutation is often used because it turns the normally brown bristles (and cuticle) yellow [4101]. Such ﬂies are useful for cell lineage analysis because any body part that develops clonally must come 1

2

IMAGINAL DISCS

3 1

2 5

4

8

9

Discs

h 7 6

10

1 2 3 4 5 6 7 8 9 10

Labial Clypeolabral Humeral Eye-antennal 1st Leg 2nd Leg 3rd Leg Wing Haltere Genital

FIGURE 1.1. Imaginal discs and their cuticular products. The ﬂy exterior is assembled from separate parts (like an automobile).

The epidermis of the head and thorax come from 9 bilateral pairs of discs (one of each kind is shown), and genitalia come from a medial disc, so there are 19 discs total. Abdominal wall comes from histoblast nests (h): tergites from dorsal nests, and sternites and pleurae from ventral nests. Discs are drawn to the same scale, and are oriented to display their mature shapes and folding. Placements are approximate. Clypeolabral and labial discs are attached to the pharyngeal skeleton (black hooks) [3285], while other discs adhere to other larval organs (not shown) [527,834,4565]. “Humeral” is synonymous with “dorsal prothoracic” disc. Bristles are omitted for clarity, and ﬂank sclerites are simpliﬁed. An adult fruit ﬂy is ∼3 mm long. Full-grown larvae are roughly twice that length [3421]. About half the larval midsection is omitted here. Adapted from [1739,4565]. Discs look more alike than the structures they produce. The same is true at the cell level, where discs are nearly indistinguishable by ordinary histology [3165,4424]. Even at the molecular level, different discs make virtually identical suites of proteins [1459,1611,3625,3756,3865], although amounts vary. The reason for these common features – as later chapters show – is that all discs use the same basic “toolkit” of molecules for intercellular signaling [662], although the circuitry (i.e., how those molecules interact) is tailored to the disc-speciﬁc patterns [1440].

from one single male or female progenitor cell and hence be purely yellow or brown. Sturtevant discovered that cuticular derivatives of all the larger discs can be bisected by a yellow/brown

boundary. Hence, these discs do not develop as clones. Subsequent studies found mosaicism in the smaller discs as well [1370, 2026, 2029, 2828]. By implication, each disc must come from ≥2 cells [2411]. In fact, when discs are ﬁrst

CHAPTER ONE. CELL LINEAGE VS. INTERCELLULAR SIGNALING

P

3

A

A y LOF y LOF

D

y+

L

P

X D

X X

R L

R V

V

isc ng d wi

D

R

wing dis c

isc ng d wi

L

D

R

wing dis c

L

V

V

fly #1

fly #2

FIGURE 1.2. The nonclonal nature of ﬂy development. The irrelevance of cell clones to pattern formation is seen in the piebald

variegation of sexually mosaic “gynandromorphs” (middle panel) [1370,2026]. Such ﬂies are typically half male (gray) and half female (black) [1715,2950]. They start life as a heterozygous female (2X) zygote but lose an X chromosome from one nucleus at the ﬁrst mitosis to create a male (1X) clone (ﬂy 1, top panel) [1695]. If the X that remains has the yellow LOF (y LOF ) allele (enlarged gray circle), then the descendants of that nucleus will make yellow (instead of brown = wild-type) bristles or cuticle in the adult (ﬂy 1, bottom panel). The two embryos at the top of the ﬁgure (A, anterior; P, posterior) differ in the orientation of the ﬁrst mitotic spindle [3274,4021]. This disparity causes the male/female boundary to trace different paths in the cuticle (middle panel) [4649,4652,4845]. The adults are bisected in the middle panel, and the cross-sections are turned ∼90◦ to a frontal view in the bottom panel (D, dorsal; V, ventral; R, right; L, left). The outer ring of circles (nuclei) schematically represents the thoracic epidermis. The inscribed “tree” represents an imaginary series of mitoses (branch points) from the initial two nuclei to the adult epidermis. Bristle numbers and cell densities are drastically reduced for clarity. If the wing disc (dashed outline) were a clone – i.e., derived from a single nucleus – then it should be purely yellow or brown because its progenitor nucleus must be one or the other. In actual gynandromorphs, however, the wing disc is often mosaic (R disc in ﬂy 1 and L disc in ﬂy 2), so it cannot be a clone. Moreover, the ability of the male/female boundaries to pass between any two landmarks (e.g., the different pairs of bristles in ﬂy 1 vs. ﬂy 2) argues that the patterns of cell lineage within the disc (inscribed trees) must also vary from ﬂy to ﬂy. Overall, therefore, such ﬂies reveal a fundamental uncoupling between pedigrees and patterning. This uncoupling is abstractly seen in the ability of the male/female “hour hands” to lie anywhere on the epidermal “clockface.” The two ﬂies shown here are only two examples from a large set of possibilities.

4

detectable histologically, each contains at least 10 cells (cf. Ch. 4). It is a quirk of history that the full import of Sturtevant’s study was only realized 40 years later [119] when Antonio Garc´ıa-Bellido and John Merriam used Sturtevant’s data to map the embryonic disc primordia [1370].

No part of a disc is a clone, except claws and tiny sense organs Yellow/brown gynandromorphs are as eye-catching as a herd of Appaloosa horses because each individual has a unique pattern of colored patches (Fig. 1.2) [2026]. Their harlequin variegation is due to (1) the random orientation of the zygote’s ﬁrst mitotic division in all three dimensions from one individual to the next [3274, 4021] and (2) the tendency of sister nuclei to stay together during cleavage [4899]. The male/female line hence intersects the egg surface at random angles [4222, 4845], and the yellow/ brown boundary should bisect any given area of the adult surface if a sufﬁciently large population is examined – unless that area is delimited clonally. Among the 96 specimens that Sturtevant analyzed, many groups of cuticular landmarks were divided by such boundaries. This “indeterminate” cell lineage was epitomized by two pairs of bristles that belong to the wing disc: the dorsocentrals and postalars. From one ﬂy to the next, Sturtevant found that both dorsocentrals may be alike [i.e. both male or both female] but different from both postalars, or the posterior dorsocentral and posterior postalar may be alike but different from the corresponding anteriors, or any one of the four may be different from the other three. Such relations occur for any group of mesonotal bristles one examines. [4180]

IMAGINAL DISCS

Indeed, male/female boundaries meander relatively freely through every bristle array on the adult surface [1800, 2026, 3007, 3539, 4652]. Clearly, discs are not balkanized into subregions where individual cells obey commands such as “Divide ‘n’ times and tell your descendants to make this part of the adult.” The only exceptions are (1) bristles and sensilla [3441] whose few component cells (≤10) come from single “mother” cells and (2) claws [1356], which follow a similar developmental path [1587]. Additional instances are found in embryonic development – e.g., neural ganglia [627], muscle subtypes [250, 3684, 3698], and cardiac precursors [1339, 4194, 4547]. Wherever cell-type determination is uncoupled from cell lineage – as here in the case of large-scale patterning within discs – it must perforce rely on intercellular signaling [293, 354, 4727].

Cells belong to lineage ‘‘compartments’’ Despite the rarity of rigid pedigrees in disc development, cells commonly obey looser edicts such as “You may make any portion of region ‘R’, but nothing outside it” [4671]. Regional limits of this kind were discovered in wing discs when marked cells were spurred to grow faster than background cells. Oversize anterior or posterior clones grew up to – but failed to cross – a boundary that roughly bisects the disc [1376, 1377], and analogous “compartments” were later found in halteres [1358, 1771], legs [1800, 2449, 4076], antennae [2931], genitalia [1107, 2028], and the proboscis [4144, 4145]. Compartments are essential for patterning (cf. Ch. 4 ff ), but their lineage constraints per se are not [754, 2428, 2448, 2677, 4491]. Hence, the existence of these clans does not negate the “Proximity vs. Pedigree Rule” [3445] enunciated above. Put simply, this rule asserts that cells select fates based on input from peers, not parents [354, 526, 1808].

CHAPTER TWO

The Bristle

Tactile stimuli are hard for arthropods to detect through the armor of their rigid exoskeleton [1666, 3582]. To solve this problem, ﬂies use bristles (Fig. 2.1). When a bristle is deﬂected, the pivoting of the shaft in its socket deforms the dendrite of a neuron attached to the shaft’s base [789, 1352, 2174, 2787]. The resulting depolarization sends an action potential to the central nervous system (CNS) [1118, 2173, 2196, 4527]. Flies can pinpoint sensations because axons from different bristles get “wired” to different CNS target cells during metamorphosis, although much remains to be learned about the topology of these neurosensory maps (cf. Ch. 6). Mechanosensory bristles are formed by 5 cells: 2 superﬁcial cells that secrete cuticle (the shaft and socket cells) and 3 subepidermal cells that do not (the neuron, sheath, and glial cells) [2475, 3351, 3552, 3832, 4531]. These 5 cells descend from a “sensory organ precursor” (SOP). The SOP divides to produce one daughter (IIa) that yields the outer cells, and another (IIb) that yields the inner cells [1447, 1741, 1925]. The sheath cell wraps the neuron’s dendrite [602, 789, 3351], while the glial cell wraps the axon [2173]. A sixth cell – the “bract cell” – is found in association with bristles on the distal leg and proximal wing [524, 1714, 1808]. It secretes a thickened hair (“bract”) that is pigmented like the bristle shaft but much smaller [3362, 3421]. The bract cell is not part of the SOP clone [1808]. The way in which it is recruited from epidermal cells is discussed later. Until 1999, the glial cell’s origin was obscure [1463, 1465, 1741, 1925], and only the shaft, socket, sheath, and neuron were considered to comprise the SOP clone. In 1999, a debate about the sequence of bristle cell mitoses [2680, 3550] prompted a reinvestigation of the mitoses themselves [1447, 3549], whereupon a new mitosis was

discovered. It had hitherto been overlooked because the glial cell is small and migrates away from its birthplace. Pre-1999 models are being revised to include this amendment [2382]. Chemosensory bristles have all the components of a mechanosensory bristle plus 4 extra neurons, whose dendrites project to a pore at the shaft’s tip [1741, 3061, 3529, 4841] where they detect chemicals (Fig. 2.8) [3835]. Strangely, such bristles (on the legs and wings at least) are also photosensitive, with independently entrainable circadian clocks [2333, 3327, 3401]. Aside from sensory modality [3005], ﬂy bristles also vary in size, shape, pigmentation, and pattern. Bristles are intriguing not only because their stereotyped mitoses violate the general rule of indeterminate lineage (cf. Ch. 1), but also because they encapsulate the problem of differentiation (how do cells acquire differences?) [2424, 2577, 4658]. In theory, the instructions for assigning fates could be unequally inherited from the SOP, with no need for cross-talk among descendants. According to this “Obey Your Mother! Model,” bristle cells adopt fates based on cues inherited from their mothers. The main cue appears to be the presence or absence of a membrane-associated protein called “Numb.” Numb has all the features expected for a heritable determinant of cell fate.

Numb segregates asymmetrically and dictates bristle cell fates The gene numb was isolated in a screen for mutations affecting the embryonic peripheral nervous system (PNS) [4417]. In a seminal 1994 article that provided the key to deciphering bristle differentiation, Michelle Rhyu et al. 5

6

IMAGINAL DISCS

SOP apical

P

A s al

mod subp ular rogr am

ba

Asymmetry

Numb crescent 0:00

Mitosis

P

A

Memory?

0

Reset

1

IIa 0

1 IIb o

90 rotation of spindle

1 2:44

0 0 0

l

10

5:00 l

10 10

101 cell

Neur o

n

ll th ce Shea

t cell Shaf

Sock

et ce

ll

100

basa

01

apica

00

cell

A l

P

11 Glial

IIIb 10

3:40

Glial

Bract cell

?

basa

l apica

Bract

1 1

Differentiation

Cuticle

Epidermal cells

To CNS

CHAPTER TWO. THE BRISTLE

in San Francisco reported that numb mutations also affect adult bristles [3579] and, more important, that Numb protein is distributed unequally during SOP divisions. Indeed, this was the ﬁrst gene product in ﬂies ever shown to segregate asymmetrically in mitosis, although others soon followed [2021]. Within the SOP lineage, 4 cells inherit Numb (IIb, shaft cell, glial cell, neuron; Fig. 2.1) [1447, 3579, 4542] while 4 do not (IIa, socket cell, IIIb, sheath cell), and mutant defects are generally consistent with this parceling. Thus, numb null mutations cause SOPs to produce 4 outer cells and no inner ones – implying that IIb adopts a IIa fate – and the outer cells are often all sockets, so a shaft-to-socket transformation must also be involved. A third conversion (neuron-to-sheath) occurs in hypomorphs [4542]. Collectively, these phenotypes imply a fate-assigning role for numb at every mitosis in the lineage, with the possible exception of the glia-producing IIb mitosis, which, as mentioned above, has only recently begun to be studied. The history of a cell’s Numb states can be denoted by the left-to-right order of digits in a binary code (Fig. 2.1), where “1” signiﬁes Numb’s presence and “0” its absence. Thus, the various sister cells in the SOP lineage would have the following paired codes: IIa (0) vs. IIb (1). Socket (00) vs. shaft (01). IIIb (10) vs. glial cell (11). Sheath (100) vs. neuron (101). From the standpoint of a strict “Coding Model,” the code would be causal. That is, a bristle cell’s fate would

7

be dictated by the series of Numb states (0 or 1) experienced by its ancestors. This code would explain the null phenotype where all cells assume a 00 (socket) state, and it would also explain the hypomorphic condition where neurons (101) switch to sheaths (100). To wit, leaky Numb levels might be high enough to let IIb attain its “1” state but not to push neurons into their later “1” state. One test of this model would be to overexpress numb. Flooding the lineage with Numb protein should raise all “0” states to “1” and cause all cells to differentiate as glia (11). When UAS-numb is driven by a Gal4 transgene expressed in SOPs, no clusters of 4 glial cells were reported [4542]. The most extreme defect was a 4-neuron trait where IIa likely became IIb (0 1) and sheath cells became neurons (100 101). Milder abnormalities were also seen, including “2 sheaths: 2 neurons” (0 1 but not 100 101) and duplicated shafts (00 01 but not 0 1). Overall, the data agree with the model, although the failure to force cells into a glial fate is problematic. Perhaps the excess Numb cannot prevent Numb’s level from being reset to “0” in IIb (Fig. 2.1). Additional support for the model comes from ﬂies carrying a hs-numb construct (numb joined to a heatshock promoter). When such ﬂies are heat-shocked around the time of SOP mitoses, they display “2 sheaths: 2 neurons” as well as “2 shafts, sheath, neuron” (socketto-shaft conversion) and “socket, shaft, 2 neurons” (sheath-to-neuron). These defects are explicable by the forced presence of Numb in the IIa (0 1), socket (00 01), or sheath (100 101) cell [3579]. Four-neuron

FIGURE 2.1. Development of a mechanosensory bristle from a sensory organ precursor (SOP). Compass (upper left) gives initial

directions (A, anterior; P, posterior). Times (hours: minutes at 23◦ C) are for microchaete mitoses on the notum but are similar for other bristles [1447]. The SOP arises from an ordinary epithelial cell. It starts to divide (at ∼16 h after pupariation) to form IIa and IIb. IIa’s daughters will make a socket and shaft. IIb’s daughters are IIIb and a glial cell. The glial cell is smaller and buds off basally in the manner of a CNS neuroblast division [1073, 1740]. IIIb divides to form a sheath cell and neuron. Some bristles have a thick hair (“bract”) atop their sockets (inset), which is made by a clonally unrelated cell. Each mitosis obeys stereotyped steps (dashed trapezoid) that comprise a modular subprogram: (1) Numb localizes to one side of the cell cortex (crescent), (2) segregates to one daughter, and (3) alters cell fate. Letting 1 and 0 signify Numb’s presence or absence, each cell can acquire a unique code if it “remembers” its former Numb states. Imaginary memory registers (underlined spaces) are shown for a few cells, with left-to-right order recording successively later states. For such a binary code to work, IIb must eliminate (“reset”) Numb before dividing. When SOPs are prevented from dividing, they become neurons [1743]. This result has been interpreted as a default condition, but it may instead reﬂect persistence of Numb: the continual presence of Numb should lead to a “nonsense” code (111) that might be interpreted as “neuron” (101). The mechanism whereby cells remember former Numb states is unknown. Timing and branching of the pedigree are as per [1447, 3549]. Other details are based on [1449, 1741, 1808, 3579]. See [3195] for lineage comparisons with other sensory organs. N.B.: Grooves are absent from some bristles (e.g., sex comb teeth [1714]). Epidermal cells are sometimes aligned with this degree of precision [2388], although they need not be. Chemosensory bristles have 4 additional neurons (cf. Fig. 2.8) [4125], and their SOPs obey a different lineage [3529]. See also App. 7.

8

IMAGINAL DISCS

?

1cleave

cleave

P

P

* Notch* Not ch

Delta

?

Su(H) Delta

2 move

P

*

Su(H)

can't move because tethered by Numb

P

3

P Su(H)

ch

* Notch* Not ch

Numb

ON

OFF

E(spl)

Nuclear Notch Model

E(spl)

Socket

Shaft

Catalysis Model ?

? Su(H)

Su(H) P

* Notch*

P

Notch Delta

* Notch* Notch

Delta

1 activate

P

P

*

Su(H)

Numb

can't activate because blocked by Numb

2

ON

OFF

E(spl)

E(spl)

Socket

Shaft

CHAPTER TWO. THE BRISTLE

phenotypes were not observed, probably because pulses were too short to affect all three rounds of (asynchronous) mitoses. If Numb were a traditional “cytoplasmic determinant,” then it would specify only one type of tissue or cell [1904]. On the contrary, it marks 4 different cells in the SOP lineage. Moreover, it plays similar roles in sense organs of the larval PNS [3579, 4417], in neuroblasts of the embryonic CNS [2451, 3579, 4028, 4523], in cardiac cell progenitors [1339, 4194, 4547], in sibling founder cells of larval vs. adult muscles [653, 3684], and in muscle subtype determination [251, 910, 3263, 3687]. Thus, its role transcends histotype. Evidently, Numb functions as a versatile switch that enables daughter cells to become different from one another, regardless of what those differences may be [831, 1761, 3263, 3579, 4875]. As a binary digit (“bit”), Numb is the best example ever adduced that ﬂies can use abstract symbols for instructions just as computers employ machine language. As explained below, this “Numb Epiphany” of 1994 is not only helping to elucidate how genes can work as switches, but it is also revealing how an intrinsic mechanism of fate speciﬁcation can dovetail with an extrinsic pathway of intercellular signaling.

Delta needs to activate Notch, but not as a signal per se Although the Numb code should be sufﬁcient for assigning all fates, some cell interactions have also been implicated. The 4-neuron trait that is caused by gain-offunction (GOF) numb manipulations is also seen with loss-of-function (LOF) mutations in Delta (Dl) and Notch (N) [1742, 3272]. Because Dl and N mediate “sibling rivalries,” whereby equivalent cells become different (cf. Fig. 3.6) [2222, 3022], they could – in theory – create binary codes by refereeing a series of bouts (winner = 1; loser = 0) without relying on cell pedigrees at all [1614].

9

Might fates be computed by either lineage (via Numb) or signaling (via Dl and N), with one agent assuring success if the other fails? No, because such redundancy would imply that phenotypes should be wild-type unless both strategies fail, but (as stated above) fates can be altered by single LOF mutations in numb, Dl, or N. Rather, it seems that the two devices are connected in series, not in parallel. Dl and N are transmembrane proteins that interact as ligand (signal) and receptor (receiver), respectively [1204, 2626]. When a N-expressing cell contacts a Dl-expressing cell, N is activated by dimerization [3022] or oligomerization [2209, 2299]. Activation causes N’s intracellular domain (“N-intra”) to detach from the membrane and go to the nucleus, where it stimulates transcription of target genes [4155]. Numb may block signaling by tethering N-intra to the cortex (Fig. 2.2), thus keeping it from reaching its targets. Enough Numb would normally be present to sequester all N, although an artiﬁcial excess of N could escape Numb’s grasp and cause the kinds of N GOF phenotypes that are seen [1307, 1651]. The need for ligand may suggest extrinsic signals, but there cannot be any instructive (on/off) signaling per se [1433] because Dl is expressed at equal levels in IIa, IIb, and surrounding nonbristle cells [3270]. Evidently, Dl plays only a permissive role, essentially like a seaman sending Morse code by using a shutter (Numb) to blink a light (Dl-N) that stays on. This “Blinker Model” supposes that Dl’s job is merely to keep N active so that the nucleus only gets a “N = off” signal when Numb is present. Mosaic analyses suggest that the SOP descendants themselves supply one another with the ligands for N stimulation, with no reliance on surrounding epidermal cells [4859]. This intrabristle cross-talk has been conﬁrmed in an interesting experiment. When Dl is overexpressed in the neuron, the adjacent shaft cell

FIGURE 2.2. Models for Notch signaling and its blockage by Numb. Black rectangles are proteins, and connecting “wires” are

binding sites. Contact with Delta ligand on a neighbor’s surface activates (asterisk) the Notch receptor, possibly by dimerization (partner outlined) [3022]. Cells that lack Numb (left) can relay the signal to its nucleus, while those that express Numb (right) cannot. The models differ in how Numb stops the signal. In the Nuclear Notch Model (above the line) [1307, 1448, 1651, 2299, 4027, 4244, 4542], Numb stops Notch from leaving its roost (ghost image) by anchoring it to the membrane [2267] via an unknown linker (“?” = possibly Partner of Numb [2609]). In the Catalysis Model (below the line) [112, 132, 1131, 3022, 4244], Numb blocks an active site for Su(H) activation (covalent modiﬁcation?). Numb is shown binding Notch at a phosphotyrosine (P), but Numb’s PTB domain is unusual and may not need a phosphate [2530, 4789], and Notch is only known to have phosphoserines [2209]. Notch resides in the apicolateral membrane [184, 1203, 1448, 2070]. The cell’s apex is carpeted with microvilli. Su(H) can activate transcription (right-angle arrow) of E(spl) (a.k.a. “m8”; cf. Fig. 2.4) by binding its promoter (gray rectangle), but E(spl) may not dictate bristle cell fates, nor is Su(H) needed for signal relay in neurons or sheath cells (see text). Estimates are that a signal at the membrane takes ∼20–90 min to cause detectable changes in target gene expression [184]. See also App. 7.

10

transforms into a socket cell (Fig. 2.7c) [2008]. Clearly, Numb’s lock on the Notch pathway can be artiﬁcially overridden by excess Dl. In contrast to the Blinker Model, the popular view has been that Numb merely biases Dl-N contests [1613, 2019, 2021, 2222, 3437], rather than being the sole deciding factor. Yuh Nung Jan and Lily Yeh Jan, who pioneered this ﬁeld, advocated this “Bias Model” but recognized an inherent paradox: because one sister cell should win every contest (with or without a Numb handicap) the numb null phenotype should be wild-type, but it is not (and the same dilemma applies in the CNS [552]). To explain why, they invoked time constraints [2020]: We think that . . . an intrinsic mechanism utilizing numb protein is superimposed on the Notch/Delta system to bias the competition. . . . We speculate that this Notch/Delta system is not sufficiently reliable to ensure that the two cells always acquire two different fates in the allotted time. (In the case of IIa vs. IIb fates, the time window is less than 2 hr.) . . . This hypothesis could explain the variable phenotype resulting from complete loss of numb function. In numb null clones some sensory bristles show the severe phenotype of having four socket cells, whereas other sensory bristles develop normally. Our interpretation is that, in the absence of numb, the Notch/Delta system still operates, but is not sufficiently reliable. . . . Some sensory bristle cells were able to finish the competition and form normal sensory bristles with four distinct fates, whereas others were unable to do so.

The Bias Model predicts that contests will end in Dlrich/Dl-poor (winner/loser) cell pairs. On the contrary, only Dl-equivalent pairs are detected in wild-type ﬂies [3270]. Rejecting the Bias Model in favor of the Numbdictated “Obey Your Mother!” Model still leaves the question of why all bristle sites in numb null clones do not have a 4-socket phenotype [2019]. Perhaps the normally dormant Dl-N rivalry mechanism has been awakened in these clones, in which case they should manifest Dl-rich/Dl-poor cell pairs (a testable prediction). Alternatively, unknown asymmetries may be augmenting Numb’s function (i.e., a partial redundancy). Either way, Numb’s control over N begs the evolutionary question: how did a heritable determinant (Numb) “hijack” an intercellular signaling pathway (Dl-N)?

Amnesic cells can use sequential gating to simulate a binary code If Numb is the bit in the bristle formula, then how do cells interpret 2- and 3-bit “words” for the various cell

IMAGINAL DISCS

types? A simple ratcheting mechanism, whereby cells count how many times they have been “1,” cannot sufﬁce because in that case “01” and “10” would be synonyms. It would seem that cells must use some sort of combinatorial code where genes aside from numb are used for recording previous Numb states. Figure 2.1 illustrates such a Coding Model. Do any known genes behave like a primary memory register – namely, their mutant alleles convert IIa into IIb (or vice versa) without switching any subsequent states? Among the genes whose mutant phenotypes connote a IIa-IIb switch, only Bearded (Brd) lacks later effects (App. 3) [2500]. Its GOF phenotype consists of neurons and sheath cells without shafts or sockets – indicative of a transformation of IIa into IIb. Thus, Brd could store the outcome of the ﬁrst mitosis. (N would turn Brd off in IIa.) Brd null mutants look wild-type [2500], but this impotence is attributable to redundant paralogs [2382]. There is another way of thinking about Numb’s mode of action that does not involve memory genes per se. To wit, Numb’s ﬁrst state (0 or 1) might simply “gate” IIa and IIb into divergent signal transduction pathways (STPs), so that the second Numb signal (0 or 1) is interpreted differently by IIa daughters (STP 1) vs. IIb daughters (STP 2). According to this “Gating Model,” genes that act only in the IIa STP should interconvert shafts and sockets when mutated, but should have no effect on neurons, sheath, or glial cells (IIb descendants), and separate sets of STP genes would operate exclusively in the IIb and IIIb sublineages. Indeed, Suppressor of Hairless appears to be a IIaspeciﬁc STP gene. Null Su(H) mutations suppress only part of the phenotype caused by numb LOF – namely, the shaft-to-socket switch but not the neuron-to-sheath switch – implying that Su(H) is only needed in the IIa lineage [4542]. This conclusion is bolstered by the ability of excess Su(H) to transform IIa (shaft-to-socket) but not IIIb daughters [200, 3827, 4542]. Su(H) is detected in both the IIa and IIIb lineages, but its level is highest in the socket cell (as are Su(H) transcripts [3826]) – a IIa daughter [1448]. Su(H) moves from the socket cell’s cytoplasm to its nucleus when N is activated [1448] – precisely the behavior expected for a messenger molecule [1269, 1307]. Su(H) can bind both to N (signal acquisition?) [1269] and to DNA sites (signal delivery?) upstream of genes in the Enhancer of split Complex [1131, 2453], which may control bristle cell fates (but see below). Thus, Su(H) has not only the phenotypic properties of a IIa STP agent, but also the histological hallmarks.

CHAPTER TWO. THE BRISTLE

When homozygous Su(H) null clones are induced near the time of SOP mitoses (3rd instar or pupal period) they transform sockets into shafts [3827, 4542], but when they are induced earlier (1st or 2nd instar) most sockets and shafts are missing and replaced by neurons and possibly sheath cells [3820]. This switching of IIa to IIb implies that Su(H) mediates not only the socket/shaft decision but also the IIa/IIb decision. If so, then the signal relays in STP 1 would be as follows, where States 0 and 1 indicate Numb’s absence or presence, “ ” and “ ” denote activation or inhibition, and “default” refers to the state of a cell in the absence of extrinsic signals: 1. Decision to become IIa vs. IIb (ﬁrst mitosis): State 0 (N = on): Dl N Su(H) {genes in Set 1} IIa. State 1 (N = off): Numb N. {Genes in Default Set 2} IIb. 2. Decision to become socket vs. shaft cell (second mitosis): State 0 (N = on): Dl N Su(H) {genes in Set 3} socket. State 1 (N = off): Numb N. {Genes in Default Set 4} shaft. Although it is easy to imagine how N could toggle cells from one set of genes to another, it is unclear why cells that do not receive a signal should switch their sets of default genes (Set 2 to Set 4) from one generation to the next. Conceivably, the latter is caused by a separate mitosis-counting mechanism. Counting of mitoses could be accomplished by a digital ratchet or an analog quantiﬁer [1880, 2762]. An example of an analog device exists in the ﬂy CNS, where descendants of the NB4-2 neuroblast apparently count mitoses of ganglion mother cells via the quantity of two homeodomain proteins (Pdm-1 and Pdm-2) whose concentration slowly falls [313, 314, 3713, 4804, 4816]. Alternatively, SOP progeny might get their timing cues from the cascade of genes that respond to ecdysone [247, 2111, 2134, 3059, 3814]. Indeed, SOP mitoses malfunction at speciﬁc macrochaete sites when ecdysone levels are depressed [3990], possibly due to this hormone’s management of disc cell cycles [1185, 1599]. It should be possible to distinguish intrinsic vs. extrinsic clocks by letting SOPs differentiate in vitro, as has been done for neuroblasts [485]. With a “schedule” strategy (i.e., events A, B, and C are triggered independently at clock times 1, 2, and 3), later steps should occur

11

when early ones are blocked; whereas with a “dominoes” strategy (A licenses B which licenses C), later steps should not proceed in the absence of earlier ones [1805, 2943, 3010]. In either case, the phenotype expected for LOF mutations in “counting genes” would be completion of SOP mitoses but no differentiation because descendants will wrongly think that they are still in the ﬁrst generation [916]. One gear in the escapement mechanism could be the p69 Tramtrack protein, which helps specify bristle cell fates (see below). Overexpression of Tramtrack during part of the cell cycle can halt bristle differentiation [3502], but it also blocks mitosis. Prospero has similar effects in the CNS [2522]. Intriguingly, LOF mutations in bazooka cause extra cell divisions in the SOP lineage [3629]. Bazooka is needed for neuroblast asymmetry and for adherens junctions [2985, 3803, 4709]. Other possible cogs in the clockwork include (1) Dacapo, a cyclin-dependent kinase inhibitor [6, 1002, 1004, 2404]; (2) Twins, the regulatory subunit of a mitosis affecting phosphatase (see below) [6, 1535, 2761]; and (3) String, a Cdc25 phosphatase that is rate-limiting for mitosis [6, 2740, 3081]. Interestingly, string has separate cis-enhancers for epidermal vs. SOP mitoses [2480]. In nematodes, some Notch-dependent fates are indeed gated by cell-cycle phases [79], and a few recently recovered ﬂy genes that are suspected of being SOP gating agents also interact with Notch [6]. Assuming that a gene in Set 2 diverts IIb into a new transduction pathway, a second messenger other than Su(H) would need to mediate the sheath/neuron decision. Su(H)-independent Notch pathways operate elsewhere [2353, 2453, 2540, 2549, 2747, 2839, 3694, 3695], so this idea is plausible [444, 4542, 4581]. In fact, a IIb-speciﬁc transducer called “Sanpodo” (Spdo) has been identiﬁed for the larval PNS, and it plays a similar role in the CNS [3985]: (1) spdo acts downstream of numb, (2) spdo null alleles transform sheaths into neurons but have minimal effects elsewhere in the SOP lineage, and (3) spdo may act likewise in adult bristle development [6, 1128, 3728]. Spdo is a homolog of tropomodulin – a protein associated with microﬁlaments. Two conjectures have been offered as to how Spdo might aid signal transduction [1128]: (1) Spdo could be part of a multiprotein complex (including N) at the cortex, or (2) Spdo could act in a cytoskeletal process that transports ligand-activated (truncated) N to the nucleus. (Both ideas ﬁnd support in Canoe, which colocalizes with N [1203, 4236] and has kinesin- and myosin-like domains that could mediate transport [2882, 3417, 3418].) Thus, STP 2 may

12

IMAGINAL DISCS

use Spdo in the IIIb portion of the SOP lineage: Decision to become sheath cell vs. neuron: State 0 (N = on): Dl N Spdo? {genes in Set 5} sheath. State 1 (N = off): Numb N. {Genes in Default Set 6} neuron. If the Gating Model is correct, then Numb would be acting less like a croupier (dealing cards that are stored by sentient cells and interpreted collectively after a full hand is dealt) and more like a gondolier (steering forgetful cells to destinations by choosing a path at each intersection).

Notch must go to the nucleus to function The issue of whether Notch acts directly in the nucleus was debated for years [1891, 2222, 3153, 4244, 4823] and has recently been settled in favor of a nuclear role [443, 446, 711, 1613, 2514], although some skeptics remain unconvinced [113, 4809] and recent data support their contention that Su(H) can act without N [218, 2255]. The history of the debate is worth recounting because (1) its grittiness shows the power of scientiﬁc dialectic and (2) its reliance on imperfect techniques shows the fallibility of scientiﬁc deduction. However, readers may safely skip this section without losing the thread of the main story. The controversy began in 1993, when N-intra (N’s intracellular domain) was found to be able to enter the nucleus and activate the Notch pathway [1271, 2542, 4161]. It was unclear whether these correlated events were causally interrelated. Based on these ﬁndings, a “Nuclear Notch Model” was formulated (Fig. 2.2). It postulated that N-intra detaches (by proteolysis) from the extracellular part of N upon binding of ligand and goes to the nucleus where it relays the signal. Why else, the model’s advocates asked, would N have nuclear localization sequences (NLSs) [132, 4161]? Skeptics stressed that N is undetectable in nuclei of wild-type ﬂies [112, 368], but defenders countered that this invisibility could be due to rapid degradation triggered by N’s PEST motif [2299, 2542]. Recent work indeed conﬁrms that (1) N-intra is needed in only small amounts to carry out its nuclear function [3805] and (2) N-intra accumulates when the proteasome is disabled [3821]. Because N has no obvious DNA-binding motif of its own, the presumption has been that N-intra functions as a “co-activator” that binds Su(H) or another transcription factor to activate downstream genes [1448, 2542].

Although now considered proven (see below), the coactivator idea is still hard to reconcile with the genetics of Su(H) and deltex: 1. The ability of excess Su(H) to transform shafts into sockets [3827] is puzzling if Su(H) needs N-intra, because shaft cells should lack nuclear N-intra (due to Numb). Conceivably, excess Su(H) compensates for the dearth of N-intra by behaving as an activator all by itself. This notion is consistent with the fact that N and Su(H) elevate transcription synergistically [750, 1131], but it is contradicted by evidence that Su(H) recruits co-repressors when N-intra is absent [449, 1330, 2129, 2941]. Perhaps, a sufﬁciently high titer of Su(H) swamps the pool of co-repressors, leaving “naked” Su(H) as the predominant species, and the solo Su(H) is then free to turn on certain target genes. Other target genes, however, are turned off, so its effects must be context dependent [1329, 1330]. 2. The gene deltex (dx) was named for the resemblance of its LOF phenotype to that of Delta – namely, wing veins that widen into deltas at their tips [2561, 4778]. It is one of only four genes recovered in an extensive screen for modiﬁers of N GOF lethality [4778, 4780]. When Deltex binds N’s ankyrin repeats, it apparently displaces Su(H), which goes to the nucleus [1448, 2746]. Such displacement implies that excess Deltex should (1) sequester N in N-Deltex heterodimers, (2) prevent N-intra from joining Su(H) as a co-activator, and (3) result in a N LOF phenotype [132]. Instead, overexpression of deltex yields a N GOF phenotype [2746]. Strangely (like Brd), deltex LOF mutations rarely affect bristle cell fates, so either deltex plays no role in the SOP clone or its function is redundantly shared by another gene [1570, 4778]. N’s ability to bind Su(H) [1269, 2747, 4244] suggested that inactive N might keep Su(H) out of the nucleus by restraining it at the membrane [1269, 1448, 4244]. Indeed, Su(H) colocalizes at the membrane with inactive N in cultured cells [1269, 1307], and movement of Su(H) to the nucleus depends on N activation both in cultured cells [1269, 1307] and in vivo in socket cells [1448]. However, the restraint idea made no sense genetically. If it were true, then deleting N should liberate Su(H) to go to the nucleus (just like activating N) [711, 1131, 1613, 2299, 2746]. In fact, N null mutations disable the pathway (App. 3). Until 1997, the Nuclear Notch Model (sans the restraint amendment) fended off criticism fairly well. Active N constructs were shown to go to the nucleus, bind Su(H), and increase transcription of Su(H)-responsive

CHAPTER TWO. THE BRISTLE

13

genes [750, 1914, 2043, 2611]. Still, it was unclear how the facts ﬁt into a causal chain in situ [4580]. In 1997, several ﬁndings were reported that seemed to contradict the model, although each challenge had a technical loophole: 1. Activated N constructs that are anchored to the membrane (in ﬂy or mammal cells) are as effective at stimulating transcription of Su(H)-dependent reporter genes as constructs that demonstrably go to the nucleus [132, 1131]. (However, proteolytic conversion of anchored N to free N-intra could not be ruled out, and minute amounts of nuclear N-intra might be enough for reporter activation.) 2. Disabling both NLSs of hNotch1 (the human Notch homolog) – by replacing 2 a.a. and deleting 49 a.a., respectively – reduces nuclear localization but does not signiﬁcantly decrease transactivation of Su(H)responsive genes [132]. (However, nuclear localization was not abolished, and residual N-intra in the nucleus may sufﬁce for reporter activation.) 3. Eliminating hNotch1’s only stable binding site for the Su(H) homolog (in its RAM23 domain [2747, 4244]) does not signiﬁcantly decrease its ability to transactivate Su(H)-dependent genes [132]. (However, Su(H) can also bind hNotch1’s ankyrin repeats, so the reduction in binding may not be enough to stop coactivation.) Doubts raised by these results made an alternative “Catalysis Model” look more attractive (Fig. 2.2) [1613]. In that model, transient contact with N supposedly changes Su(H) to an activated state, and “Su(H)*” then enhances transcription of downstream genes with no further need for N [112, 1448, 1651, 2299, 4244]. Because N itself has no known enzymatic activity [1613], N might use a binding partner as the catalyst [1131]. Conceivably, N alters Su(H)’s shape to unveil a domain without covalent modiﬁcation [132, 1131]. This “hit-and-run” scenario agreed with (1) the instability of N-Su(H) binding and (2) the fact that N and Su(H) do not always colocalize [4244]. In 1998, the tide turned for the last time. Incisive studies provided direct evidence for the Nuclear Notch Model. They showed ligand-dependent proteolysis and transit of N-intra to the nucleus, as well as transcriptional stimulation by amounts of nuclear N below histological detection [443, 711, 2514, 4582]: 1. Upon binding of ligand, mNotch1 (the mouse Notch homolog) is cleaved on the cytoplasmic side of its transmembrane domain – between residues that in

2.

3.

4.

5.

the ﬂy correspond to Met-1762 and Val-1763 [3805]. Mutating the Val to Leu or Lys virtually blocks cleavage, but even the residual amount of cleaved product is able to activate transcription of a reporter gene. The quantity of nuclear N needed for such activation is so small that it is barely detectable immunologically, even when sensitivity is enhanced with multiple Myc tags on N-intra. This result explains why nuclear N had hitherto been “invisible” in vivo, and it validates the idea that trace amounts of cell-surface receptors can directly inﬂuence nuclear events [2030, 2031]. Inserting a Gal4 DNA-binding domain into the intracellular tail of full-length N produces a receptor that can activate UAS-regulated reporter genes in a ligand-dependent manner in situ [2454, 4155], whereas similar insertions into the extracellular domain do not. Hence, cleavage and nuclear transit of N-intra are natural consequences of ligand binding. Inserting domains for transcriptional activation (Gal4 or VP16) or repression (WRPW or an Ala-rich piece of Engrailed) into N’s tail, respectively, rescues or fails to rescue N null embryos – implying that N-intra directly (in the nucleus) regulates targets of the Notch pathway in situ [4155]. A cleaved, Su(H)-bound form of N that is soluble and phosphorylated was identiﬁed in ﬂy embryos in addition to full-length N [2211], and the ratio of processed to full-length N was shown to depend on (1) the presence of N’s binding domain for Dl and (2) the amount of Dl both below and above the wild-type level. The cytoplasmic domain of N was found to have an intrinsic ability (85% that of Gal4) to activate transcription at a heterologous promoter in yeast and in vivo [2211]. To conﬁrm that N is a co-activator for Su(H), a foreign activation domain was substituted for N. When VP16 was fused to Su(H), the Su(H)-VP16 product activated transcription of m8 (a natural target of Notch signaling) in N null embryos [2211].

The inescapable conclusion is that a piece of the N molecule stimulates transcription directly in the nucleus after ligand-dependent cleavage of full-length N. The implied stoichiometry of “1 N molecule: 1 unit of activation” is consistent with the dose sensitivity of the Notch pathway [118, 1797]. This situation contrasts with other transduction pathways, where the quantity of second messenger is catalytically ampliﬁed relative to the input signal [3297].

14

Cleavage of precursor (in Golgi)

binds Scale: 100 a.a.

1 LNG repeats (3x)

Ligand-induced cleavages

2 3 N-intra

C1693 C1696

Key:

NLS (2x)

EGF-like repeats (36x)

leader 11 12

24

NH2

PEST

ankyrin repeats (6x)

opa

Notch

29

COOH

*

Delta Fringe

*

RAM23

Dsh

Serrate NH2

2703 a.a.

COOH

*

*

Notch

Su(H) Deltex N

C

Numb PTB

556 a.a.

CHAPTER TWO. THE BRISTLE

How does Numb ﬁt into this scheme? An obvious possibility (stated above) is that Numb stops Notch signaling by tethering N-intra to the cortex, and Numb does bind N-intra in vitro [1307, 1651]. If this were the whole story, then freeing Numb from the cortex should let N-intra function in the nucleus (hence shifting State 1 cells to State 0), but as the following experiments demonstrate, this prediction is not fulﬁlled. 1. Deleting Numb’s “mooring cleat” (at its N terminus) releases it into the cytoplasm but does not affect its ability to switch cell fates (by inhibiting N) when overexpressed [2269]. 2. The mouse protein “Numblike” resembles Numb except that it is cytoplasmic [4876], and expressing a Numblike mouse transgene in numb null ﬂies shuts off the Notch pathway, despite Numblike’s freedom from the cortex. 3. Numb can suppress N in cultured cells even when both overexpressed proteins are cytoplasmic [1307]. Indeed, Numb appears to block N function by sequestering N-intra in cytoplasmic heterodimers, thus keeping it out of the nucleus. While Numb’s normal function could still be to tether N, it must be able to stiﬂe N by other means. N’s Numb-binding domain contains a nuclear localiza-

15

tion sequence (Fig. 2.3), so Numb could block nuclear entry of N-intra by masking this site. Alternatively, because N’s Numb- and Su(H)-binding sites overlap [1307, 1651, 2747, 4244], Numb-N heterodimers may be unable to dock with Su(H) even if some of them enter the nucleus. (Numb does not bind Su(H) directly [4542].) A third possibility is that Numb obstructs access of the liganddependent protease [4523]. One clue to this mystery is that microtubules must be intact for Numb to muzzle N [4523]. In summary, the physical basis for the “Numb N” and “Dl N Su(H)” interactions appears to be as follows. Numb masks one or more of N’s functional domains. In Numb’s absence, binding of Dl to N releases N-intra from the membrane by proteolytic cleavage, allowing N-intra to enter the nucleus, where it functions as a co-activator with Su(H) or other transcription factors to modulate the expression of downstream genes.

E(spl)-C genes are Su(H) targets but play no role in the SOP lineage In vitro, Su(H) protein binds the nucleotide heptamer GTGG/A GAA (where “G /A ” means a G or A at this position) [492, 1131, 2453, 4408]. This heptamer resides in the promoters of at least 4 of the 13 genes in the Enhancer of split Complex (E(spl)-C) at 96F11–14 – namely, mγ , m4, m5,

FIGURE 2.3. Notch, its domains, and some of its binding partners.

Part of a cell is shown (gray = cytoplasm; microvilli at top) with two Notch (middle) and one Numb molecule (below) drawn as bars (cf. scale at upper left). Domains (variously shaded or hatched) are mapped (cf. App. 1). Other proteins (black rectangles) are not to scale. Partners are linked by hooks (cf. key), and binding sites are delimited by dashed ovals or half ovals. Despite their regularity, the various EGF-like repeats play different roles [459, 4601, 4823], and no two are identical [2182, 2542]. Delta and Serrate both bind EGF-like repeats 11 and 12 [459, 985, 3544], although they also rely on repeats 24–26 (not shown) [2419], and binding alone may not sufﬁce for activation [1943, 1944]. Fringe (cf. Ch. 6) binds repeats 24–29 [990] and at the LNG domain [2096]. Scabrous requires repeats 19–26 for its association with N (not shown) [3456], although any binding must be indirect [4601]. The famous allele split (whence the link to the E(spl)-C was deduced [3028]) is due to a missense mutation (Ile-to-Thr) in repeat 14 ts1 has a missense mutation (Gly-to-Asp) in repeat 32 (a.a. 1272) [4779]. [1747, 2182], and the widely used heat-sensitive allele N Notch is cleaved at three sites (zigzag lines) [491, 2995, 4809]. Cleavage at site 1 occurs during maturation in the trans-Golgi along the secretory pathway [368, 2299], and the fragments stay together, possibly by disulphide bridges at C1693 and C1696 [3153] (but see [446, 578]). Cleavage at site 2 occurs after ligand binding [491, 2995], releasing an ectodomain that is “swallowed” by the ligand-bearing cell [2263, 3271] (cf. the reverse with Boss [601, 2318, 4211]). The smaller size of the extracellular vestige (now <300 a.a.) stimulates a Presenilin-dependent protease to make the next (ﬁnal?) cut [3533, 4156, 4582]. Cleavage at site 3 occurs in the transmembrane domain (a.a. 1746–1765) between M1762 and V1763 [2995, 3805], releasing an intracellular fragment (N-intra) that goes to the nucleus and turns on target genes as a co-activator with Su(H) [4155] (see text for evidence). For review, see [2994]. Numb, when present, apparently tethers N-intra to the membrane (cf. Fig. 2.2). Half of Numb’s PTB domain binds Notch at RAM23, where Su(H) also binds [1307, 1651, 2747, 4244], and at a less crucial C-terminal site [1139, 1307, 1651]. Deltex may displace Su(H) from Notch when Delta is absent [112, 1448, 2746, 3022], although binding sites for Deltex and Su(H) do not overlap [2747, 4244]. (Deltex switches cell fates when overexpressed but has no LOF effect on bristles [2746].) Notch is thought to dimerize via cysteines (C1693 and C1696?) [2209, 2542] but may also do so via its ankyrin repeats [2747] or opa motif (links not shown) [3353]. Binding of Dishevelled (Dsh) to the C-terminal tail [151] short circuits the Notch and Wingless signaling pathways [356, 3690]. Notch’s other binding partners include Wingless itself [4601] (see App. 3 for other modulators). Sites of phosphorylation [368, 2209, 2211, 4582] and glycosylation [2070, 4611] are not shown. Fringe is presumed to glycosylate Notch at O-linked fucose sites in EGF-like repeats 3, 20, 24, 26, and 31 [2904]. Adapted from [368, 446, 2210, 2542, 4611]. See [4602, 4603] for apparently heretical modes of Notch signaling. See also App. 7.

16

IMAGINAL DISCS

a

E(spl)-C

-30 kb

m

-20

m

m

* * *

b70 kb

60

y

-10

m

m1 m2

50

40

M3 (7-69) M5 (14-74) M7 (9-69) M8 (6-66) M β (9-71) M γ (11-73) M δ (11-73)

30

sc

*

m3 m4

*

20

* **

10

0

l’sc

-10

helix 1

-20

-30 kb

ase T1

T2

* *

basic

20 kb

10

m5 m6 m7 m8 gro

AS-C

ac

c

0

*

loop

helix 2

KTYQYRKVMKPLLERKRRARINKCLDDLKDLMVECLQQEGEHVTRLEKADILELTVDHMRKLK KTQHYLKVKKPLLERQRRARMNKCLDTLKTLVAEFQGDDA--ILRMDKAEMLEAALVFMRKQV KTYQYRKVMKPLLERKRRARINKCLDELKDLMAECVAQTGD--AKFEKADILEVTVQHLRKLK KTQIYQKVKKPMLERQRRARMNKCLDNLKTLVAELRGDDG--ILRMDKAEMLESAVIFMRQQK KTYQYRKVMKPMLERKRRARINKCLDELKDIMVECLTQEGEHITRLEKADILELTVEHMKKLR KTYQYRKVMKPMLERKRRARINKCLDELKDLMVATLESEGEHVTRLEKADILELTVTHLQKMK KTQHYRKVTKPLLERKRRARMNLYLDELKDLIVDTMDAQGEQVSKLEKADILELTVNYLKAQQ

conserved:

Hairy (27-89)

PLKSDRRSNKPIMEKRRRARINNCLNELKTLILDATKKDPARHSKLEKADILEKTVKHLQELQ 6

13

d

e WRPW

WRPW

Gro

CACnAG

Su(H)

CAnnTG

+ ? + + f TGTGAGAAACTTACTTTCAGCTCGGT TCCCACGCCACGAGCCACAAGGATTGTCCTCCGTCCTACGAAGTTGCAGCTGT C g

S

-700 bp

-600

NE

-500

S?SNN E

-400

-300

-200

m8 -100

+1 bp

CHAPTER TWO. THE BRISTLE

and m8 (Fig. 2.4) [171, 2453]. In vivo, reporter genes attached to these promoters are activated by Su(H), and the activation requires the heptamers [171, 2453]. The implication is that E(spl)-C genes are targets of Su(H) during Notch signaling (i.e., Dl N Su(H) “Transcribe E(spl)-C genes”). Indeed, these same promoters are responsive to activated N in vivo [171]. Some connection between E(spl) (a.k.a. m8) and N had long been presumed because a mutant allele of E(spl) enhances the phenotype of split – an allele of N (hence the gene’s name) [3028, 3896]. Seven genes in the E(spl)-C contain a “basic helixloop-helix” (bHLH) motif (Fig. 2.4) [2246, 2274]. For bHLH proteins in general, the “basic” domain mediates DNA binding and the helices mediate dimerization [1152, 2634]. Some bHLH proteins form homodimers, others form heterodimers with preferred partners, and still others can do both [68, 245, 2039, 3028, 3029, 3375]. A separate cluster of four bHLH genes – the achaetescute Complex (AS-C) at 1B1–7 – also governs bristle development (cf. Ch. 3), and certain proteins from the two complexes can heterodimerize [1484, 3171]. Whereas AS-C bHLH proteins have canonical bHLH features [348, 1152,

17

3375], E(spl)-C bHLH proteins are unorthodox in several respects (Fig. 2.4) [135, 3171, 4318]:

1. Their dimers typically bind an “N box” consensus nucleotide sequence CACnAG (where “n” denotes a variable nucleotide), instead of the CAnnTG “E box” that characterizes other bHLH proteins. Nevertheless, E(spl)-C dimers can compete with AS-C dimers at E boxes [2054], and ﬂanking bases are also relevant. 2. Their basic domain contains a conserved proline, although substituting a different amino acid does not alter their afﬁnity for N boxes [3171]. 3. Their carboxy terminus invariably ends with the sequence WRPW (tryptophan, arginine, proline, tryptophan). These features deﬁne a distinct bHLH subfamily that also includes the genes hairy and deadpan [331, 1017, 2274, 3697, 3759]. The WRPW tetrapeptide binds the ubiquitous protein Groucho [2064, 3278] – a “co-repressor” (i.e., an inhibitory factor that “piggybacks” on DNA-binding proteins). Groucho reduces transcription by recruiting a histone deacetylase [737]. Interestingly, the groucho gene resides in the E(spl)-C. By bringing Groucho to N boxes,

FIGURE 2.4. Clusters of bHLH genes and the roles of their proteins as transcriptional regulators.

a, b. Enhancer of split (E(spl)-C; a) and achaete-scute (AS-C; b) complexes. Genes are depicted as arrows, indicating the direction of transcription. E(spl)-C spans ∼50 kb on the 3rd chromosome; AS-C spans ∼100 kb near the tip of the X. Asterisks mark genes whose products have a bHLH motif. Oddly, all these genes are devoid of introns. Within each complex, the bHLH genes exhibit partial functional redundancy [2548, 4674]. AS-C bHLH proteins are transcriptional activators, whereas E(spl)-C bHLH proteins are repressors. Half circles mark E(spl)-C genes that belong to a separate (“Bearded”) gene family [2383]. c. Amino acids in bHLH motifs of E(spl)-C proteins and Hairy (AS-C proteins not shown; for code, see App. 1). Dashes are inserted to aid alignment [2274]. Numbers are residues counted from the N-terminus. E(spl)-C bHLH proteins are ∼200 a.a. long, with ∼60 a.a. in their bHLH domain. The “conserved” row pertains to E(spl)-C proteins: ﬁlled circles (invariance); unﬁlled circles (>50% but <100% identity); +s or −s (charged residues). The many +s deﬁne the “basic” domain, which, in this subfamily, has proline at position 6 and arginine at position 13 [3179]. d, e. bHLH proteins dimerize via helical domains (striped), adopt a scissors shape, and bind DNA via basic domains (black). Within each subunit, the upper and lower helices touch (unlike in this cartoon). d. E(spl)-C bHLH dimers bind an “N box” consensus sequence “CACnAG” (“n” is a dimer-speciﬁc nucleotide) [3171, 3759], which Hairy can also bind, although it prefers CACGCG [3179, 4451]. Their C-terminal “tails” (top) end in “WRPW” (as does Hairy’s [4524]) [2274, 3697], which recruits Groucho (Gro, black rectangle). Gro is a co-repressor (X’d arrow) [2064, 3278] that in turn binds a chromatin-modifying histone deacetylase [737], although E(spl)-C proteins also have Gro-independent effects [3029]. The gro gene resides in the E(spl)-C (a). e. Most other bHLH dimers (including AS-C) bind an “E box” hexamer “CAnnTG” [348, 3375], do not end in WRPW, and activate transcription (cf. the bHLH-PAS subgroup [4022, 4612]). f, g. Promoter region of the m8 (a.k.a. E(spl)) gene. f. Details. Binding sites (S = Su(H); E = E box; N = N box) are boxed (f) or shown as bars (g). “?” denotes a hexamer (function unknown) found between invertedly repeated “S” sites [171, 3075], whose interrepeat distance is constant among promoters. +s and −s signify stimulation vs. inhibition of transcription. g. “Wide-ﬁeld” view of the m8 cis-enhancer region. Dashed rectangle marks the section shown in f – viz., bases −133 to −211 b.p. from the transcription start site (“+1 b.p.”; right-angle arrow). Negative feedback of m8 onto its own N boxes [871] may help to stabilize output and minimize noise [262, 1383, 3347]. The crowding of binding sites suggests steric competition or “quenching” [1984] that mediates “either/or” (vs. “both/and”) logic [2350]. Additional N boxes and “S” sites (of varying afﬁnity) map in the 700 b.p. span (g) [2317, 3075], although we do not know whether they are all needed [449]. The base at −185 (f) may be T [171] or C [2246]. Cis-regulatory regions for all E(spl)-C genes (except gro) have been analyzed similarly [871, 3075]. Maps of loci were compiled from Fig. 3 of [3806] amended as per [2383,4767] (a) and from [636, 1538] (b). Sequences in c are from [2274]. Panels d and e are based on [1152, 2634], and panels f and g are adapted from [171, 2246, 2317, 3171]. See [2054] for an exegesis of E vs. N boxes and an exploration of target gene preferences for AS-C vs. E(spl)-C proteins. See also App. 7.

18

IMAGINAL DISCS

E(spl)-C bHLH proteins should repress the transcription of any genes that have N boxes in their promoters. One such gene is m8, which hence should repress itself, although the proximity of its N boxes to Su(H)binding sites (Fig. 2.4g) suggests that autorepression is prevented by competitive binding during Notch signaling [171]. If so, it seems odd that SOPs express a lacZ reporter linked to an m8 promoter (ectopically) when its Su(H)-binding sites are deleted [2453], but the promoter could still respond to AS-C bHLH proteins that bind its E boxes [171, 3171]. Phrasing the logic at m8 promoter as an imperative, the rule would be roughly as follows: “Turn on IF you are occupied by Su(H) or AS-C proteins, but NOT IF there is too much of your own gene product around, in which case you should remain off.” Another way of thinking about the output of m8 is as an equation whose inputs – Su(H), AS-C, and M8 – have weighting factor coefﬁcients. This glance at m8 serves to show (in microcosm) the intricacy of the control system. We do not yet know which (if any) E(spl)-C genes are expressed in the SOP progeny [991]. Genetic analysis has been difﬁcult [2561, 4885] due mainly to the functional redundancy of the bHLH genes [991, 1017, 2548, 3806]. Indeed, deletion of m8 (whose neomorphic E(spl)D allele had long implied a key role for this gene [4318]) has no detectable phenotypic effect [1018, 3806]. Overexpression of wild-type E(spl)-C bHLH genes can cause various changes in cell fates [2317, 4256], but these abnormalities may be misleading because some of them persist when conserved domains (bHLH and WRPW) are deleted [68, 1475]. The biologically relevant question is: Do E(spl)-C bHLH proteins normally assign fates in the bristle organ? Given the following data, the answer seems to be “No”:

sion of the chimeric transgene (via Gal4-UAS control) increases bristle density, but fails to switch cell fates in the bristles themselves [2063], implying that m7’s downstream targets are irrelevant to the SOP lineage. 4. Overexpressing the bHLH genes mδ, mγ , or m7 suppresses SOP initiation at many sites, but the remaining bristles have a normal set of constituent cell types [3027]. When either of the non-bHLH genes mα or m4 is overexpressed, it increases bristle density (by inhibiting bHLH E(spl)-C genes) but fails to alter the SOP lineage [92].

1. Deletion of all 7 E(spl)-C bHLH genes in somatic cell clones increases the density of bristles (for reasons discussed in Ch. 3), but has virtually no impact on bristle cell identities [991, 3027]. 2. Deletion of various E(spl)-C bHLH genes rescues the bristle-loss phenotype of null mutations in Hairless (an antagonist of the Notch pathway, see below), but does not alter the shaft-to-socket transformation also caused by these mutations [197]. 3. Replacing the WRPW (Groucho-binding) site of M7 with a transcriptional activator produces a chimeric M7 protein that should bind N boxes (since it still has its bHLH domain) but activate (not inhibit) its gene targets (due to the C-terminal substitution). Expres-

The transcription factor Tramtrack implements some cell identities

Only one bHLH gene remains a viable candidate for the setting of cell fates within the bristle organ during normal development – the AS-C gene asense. Asense is found in SOPs, and its expression persists into IIa and IIb, although no asense mRNA has been detected beyond this point [438, 1079, 2039]. Given that all SOPs make Asense, it is surprising that only one group of bristles is affected when the gene is deleted: a row of stout bristles along the wing margin [2039]. In asense null ﬂies, this row manifests empty sockets, missing bristles, stunted shafts, and twinned shafts. Twinning could indicate a socket-to-shaft or IIb-to-IIa conversion, although neither is certain because the SOP lineage here (in wildtype ﬂies) can violate the rule of ﬁxed fates (e.g., socket and shaft cells do not need to be sisters) [1741]. When the asense deletion also removes certain AS-C enhancers, a sheath-to-neuron switch is seen in the giant sensillum of the radius [1079]. The importance of asense inferred from this LOF data is not upheld by the GOF data. Overexpression of Asense (via a heat-shock promoter) induces extra bristles, but it fails to alter any cell fates within the bristles themselves [438, 1079].

The gene tramtrack (ttk) was recovered in screens for proteins that bind enhancers at the ftz pair-rule locus [502, 1731]. Its transcripts are spliced to produce two protein isoforms – “p69” (69 kD) and “p88” (88 kD) [3537]. Both isoforms typically function as transcriptional repressors [503, 1483, 4596, 4773], but p69, unlike p88, becomes an activator in late eye development [2391]. Both proteins have an N-terminal “BTB” (protein interaction) domain and two DNA-binding zinc ﬁngers, but the ﬁngers of p69 and p88 differ in amino acid sequence (and hence target DNA sequence) because they come from different exons (Fig. 2.5) [3537].

CHAPTER TWO. THE BRISTLE

a3 kb

19

tramtrack gene 4

5

6

7

8

9

10

ZnFs

A

B

A

B

11

12 kb

ZnFs

C

p69 D

p88

b

p69 (ZnFs): YRCKVCSR-----VYTHISNFCRHYVTSHKRNV-KVYPCPFCFKEFTRKDNMTAHVKIIHKI p88 (ZnFs): YRCTECAKENMQKTFKNKYSFQRHAFLYHEGKHRKVFPCPVCSKEFSRPDKMKNHLKMTHEN conserved:

Asn

p69 protein

' ' 5-ATCCT-3

d

H

C C

N

H

O

H H

C N

Adenine

N

N C

C N

C N

C

H

ine

H

ym

' ' 3-TAGGA-5

H

Th

c

YR K RNVKVYP C HS KI P C K T H Zn F I V V Zn I C C HY K F HV S R K A R C E T V F F M Y N T N T S R KD H I

H

FIGURE 2.5. Alternative splicing of tramtrack yields different DNA- binding proteins.

a. Transcripts (fragmented arrows) of the ∼9-kb gene tramtrack (ttk, 100D) encode two proteins, each of which has different zinc ﬁngers (“ZnFs”) [3537, 4773]. Kinked lines are spliced-out pieces, and lightly shaded bars are untranslated sequences. The 69-kD “p69” isoform (641 a.a.) has sections A, B, and C. The 88-kD “p88” isoform (811 a.a.) has sections A, B, and D. Shared segments (A, B) encode a “BTB” protein-interaction domain [211, 1516, 4891], and unshared ones (C, D) encode zinc ﬁngers. b. Amino acid sequences of the ﬁngers. White letters are Cys and His residues that coordinate Zn2+ ions, dashes are gaps to aid alignment, and black circles mark identical residues. c. Zinc ﬁngers (shaded) of p69 are drawn ﬂat, although each outlined half is actually an α-helix, and the other half forms β-strands. p69 binds DNA as a monomer [1186], inserting the α-helices of its ﬁngers consecutively into DNA’s major groove. Arrows indicate a.a.-base contacts. The DNA oligomer used for structural analysis was devised from p69’s enigmatic set of binding sites [1186]. p88’s ﬁngers (not shown) bind other sequences [3537, 4775] and have not been studied crystallographically. Transcriptional repression (lines radiating to X’d arrow) might be due to a C-terminal domain (BTB is N-terminal). In some unknown way, p69 (but not p88) is converted from a repressor to an activator in eye development, where the two isoforms act differently in different cell types [2391, 2529]. Both isoforms are regulated by proteolysis [1051, 2529, 4249]. d. Hydrogen bonds (shaded) between asparagine and adenine (cf. N-to-A arrows in c) vs. bonds within the A-T base pair (at right) revealed by crystallography [1187]. Such interlocks have fueled hopes for a “ﬁnger code” that could allow ﬁngers to be designed for desired DNA sequences [296, 2265]. This diagram is adapted from [4773] (a), [3537] (b), [1187, 2123] (c), and [296] (d).

20

This use of alternative splicing to send transcription factors to different gene destinations is a clever sort of grammar in cell programming [345, 2599, 3996]. The imperative “Turn off!” (or in some cases “Turn on!”) can thus act on various genes via different “Go to” addresses (the ﬁngers of p69 vs. p88) [4339]. This “Finger Shufﬂing Trick” is used by at least two other ﬂy genes: (1) the “pufﬁng cascade” gene Broad-Complex, whose BTB domain is spliced to one of four pairs of zinc ﬁngers [247, 248, 1044, 2974] in a spatially regulated manner [458], and (2) a (nonBTB) chorion transcription factor [1519, 1918]. Alternative splicing is also used for other types of “Go to” commands, which (1) add or delete NLSs that send proteins to the nucleus vs. cytoplasm [957, 2032], (2) insert a peptide that sends proteins to intercellular junctions [4574], and (3) may even direct the wiring of sensory axons to speciﬁc CNS neurons [4763]. Deleting ttk function in somatic clones causes the same 4-neuron trait seen in N LOF ﬂies, but overexpressing ttk (via a heat-shock promoter) only rarely yields the 4-socket trait of N GOF [1650]. Instead, the major GOF phenotype is “2 shafts, 2 sockets” [1650, 3502] – indicative of a conversion of IIb into IIa. (Strangely, the GOF phenotypes for p69 and p88 are similar despite their different binding-site preferences.) Hence, ttk may implement the IIa/IIb decision. Its null phenotype suggests that ttk also mediates the sheath/neuron decision because two switches must occur for an SOP to make 4 neurons (IIa-to-IIb and sheath-to-neuron). Consistent with this reasoning, p69 is detectable in IIa (not IIb) and in sheath cells (not neurons) [3502]. The rarity of 4-socket or 4-shaft defects implies that ttk plays no role in the shaft/socket decision – a conclusion bolstered by Ttk’s presence in both shaft and socket cells [3502]. Thus, as a rule, ttk is expressed in non-neural cells (or their ancestors) in the SOP lineage [3502]. Similar rules govern ttk function in the CNS and eye, so ttk may be a generic repressor of neural fates [1483, 4773]. Ttk may be kept off in IIb by proteolysis [3550] because (1) in the eye Ttk is targeted for degradation when it binds Seven in absentia (Sina) and Phyllopod (Phyl) [4249], and (2) LOF mutations in sina and phyl cause doubled bristles [671, 717] that suggest a IIb-to-IIa conversion [3550]. If ttk toggles cell fates in the IIIb sublineage (sheath vs. neuron) in the same way that Su(H) behaves in the IIa sublineage (socket vs. shaft), then is Ttk the counterpart of Su(H)? Probably not, because Ttk has not been shown to bind N (whereby it could form a factor-cofactor com-

IMAGINAL DISCS

plex). Other candidate transducers for the IIIb sublineage have been recovered [6].

Hairless titrates Su(H) Given the involvement of both Su(H) and Ttk in the IIa/IIb decision, but their separate roles in the IIa and IIb sublineages, there may be at least three transduction modes. Hairless (H) [200, 2659] also ﬁts into this scheme. Until 2000, its effects were thought to be conﬁned to the socket/shaft decision, but a detailed LOF-GOF analysis proved its participation in all three decisions [3027]. Removing H forces cells into State 0, while overexpression forces them into State 1 (App. 3). STP 1 : Decision to become IIa vs. IIb: State 0 (N = on): Dl N Su(H) {ttk, etc., but not E(spl)-C} IIa. State 1 (N = off): Numb N. H and {genes in Default Set 2} IIb. STP 2 : Decision to become socket vs. shaft cell: State 0 (N = on): Dl N Su(H) {genes? [not ttk or E(spl)-C]} socket. State 1 (N = off): Numb N. H and {genes in Default Set 4} shaft. STP 3 : Decision to become sheath cell vs. neuron: State 0 (N = on): Dl N Spdo? {ttk, etc., but not E(spl)-C} sheath. State 1 (N = off): Numb N. H and {genes in Default Set 6} neuron. Moderate H LOF alleles delete shafts (hence its name), either by blocking SOP initiation (“bristle loss”) or by converting shaft cells into socket cells (“double-socket” phenotype) [198]. Macrochaetes (large bristles) are affected more than microchaetes (small bristles), and certain sites are consistently affected more than others (Fig. 2.6a) [997, 2690]. Why should similar bristles differ in their need for the same gene product? This “Nonequivalence Riddle” has nagged theorists not only for H LOF [3053, 3054] but also for other “missing bristle” mutants [3405] – especially scute LOF whose subpatterns are allele speciﬁc [650, 767, 1108, 1328, 4100] – and for “extra bristle” mutants [733, 3028]. Topological models (wherein particular genes control particular bristles in 1:1 correspondence) were discounted because (1) phenotypes are so easily modiﬁable by temperature or overcrowding [767--769, 1997, 1998, 3405] and (2) double-mutant combinations can remove bristles outside the areas affected by either mutant alone

CHAPTER TWO. THE BRISTLE

[3405, 4187]. For the AS-C, the spatial heterogeneity is due to each bristle’s reliance on the sum of (functionally redundant) Achaete and Scute, whose amounts are set by position-speciﬁc enhancers (cf. Ch. 3). Hairless may likewise have a partially redundant “partner” in some body regions because leg bristles remain virtually normal even in H null mutants [198]. Although the reason for H ’s spatial nonuniformity is unclear, the basis for its quantitative effects is understood, thanks to a peculiar feature of this locus – namely, its “haplo-insufﬁciency.” Only 21 genes in the ﬂy genome (aside from Minutes) manifest haplo-insufﬁciency (i.e., display a mutant phenotype when present in haploid dose as a deﬁciency/+) [118]. Presumably, the products of such genes are “limiting” for the rate of particular reactions [117] or for the stoichiometry of particular partnerships [2582, 3458]. In the case of Hairless, the haplo-insufﬁciency is attributable to a physical coupling between the H and Su(H) proteins. As the name indicates, Suppressor of Hairless LOF mutations suppress H LOF phenotypes. The suppression can be mimicked by lowering the dose of Su(H). Thus, heterozygous H LOF/+ ﬂies that are also heterozygous for a Su(H) deﬁciency look nearly wild-type [117]. Conversely, increasing the dose of Su(H) proportionally enhances the phenotype of H LOF/+ ﬂies, as expected for a stoichiometric interaction (Fig. 2.6c). This titratable antagonism is illustrated most dramatically by the creation of a H LOF phenotype in a wild-type (H +/H + ) background by simply raising the dose of Su(H) to 8 (from its normal 2) [3827]. The identity of the Su(H) “substitute” that H opposes in STP 3 (sheath cell vs. neuron choice) remains to be determined [3027]. H is detectable in all SOP descendant cells [3027], but subtle differences in its levels may still exist in State 0 vs. State 1 cells. Alternatively, H may be modiﬁed posttranscriptionally by auxiliary factors in one or the other cell state. Hairless is a weird protein. It is extremely basic (overall pI = 9.5; pI in parts = 11, based on a.a. composition; Fig. 2.6d), although it has no known DNA-binding motif. It is 40% alanine, serine, or proline [200, 2657]. Its function in ﬂies seems to be to stiﬂe Notch signaling during bristle development (SOP lineage and SOP initiation) [3027, 3824], but less so during the other processes where Notch is deployed [113, 444]. Su(H) and H bind each other in yeast two-hybrid assays [171, 1332, 2657], and their interfaces have been identiﬁed [492, 2695]. Because the H- and DNA-binding domains within Su(H) overlap, H might block Su(H) by masking its DNA-binding site [492]. How-

21

ever, truncated H transgenes that contain the Su(H)binding domain do not fully rescue H phenotypes in vivo [2657], so H must do more than just dock with Su(H) [1329, 2658]. Indeed, it has recently been shown that the Su(H)-H complex binds DNA and that H recruits the co-repressor “dCtBP” (see Fig. 3.12e and [2129] sequel). Why should any antagonist for Su(H) be needed – titratable or otherwise? Shouldn’t shaft cells have a silent Notch pathway after Numb has gagged N? Perhaps, but the pathway may not be silent enough. Residual N-intra or Su(H) might linger from the mitosis that created IIa. H’s role could be to “mop up” excess Su(H) to ensure that prospective shaft cells do not hear any N signal whatsoever and hence become shafts with 100% ﬁdelity. H could set a constitutive threshold above which Su(H) must rise in order for a cell to become a socket [197, 3826]. A similar damping step has been proposed for gating N-Su(H) heterodimer entry into the nucleus [2211]. In principle, thresholds are an easy way to convert messy (analog) signals into discrete (digital) states [3573, 4513] – as Charles Plunkett, a pioneer of bristle research, argued in 1926: The only way in which I can conceive of an ‘‘all-or-nothing’’ reaction, such as the production of a bristle, being determined by the concentration of a continuously varying substance, is that the reaction occurs if, and only if, that concentration equals or exceeds a certain ‘‘threshold’’ value. [3405]

Thresholds also help to explain “ﬂuctuating asymmetry” [2693, 2901, 4460], where the left and right sides of single individuals show different phenotypes. Such asymmetries are rare in wild-type ﬂies [2866, 4746, 4747] but common in mutants such as H LOF [1961, 3405, 3571, 3572, 4514, 4674]. For H LOF , the reason may be that the amount of H sinks to the level of Su(H) in some prospective shaft cells. In the close contests that ensue, the outcome will depend on random local ﬂuctuations (a.k.a. “noise” [1383, 2259, 2518, 4513]) in both values (cf. error bars in Fig. 2.6e). Thus, a prospective shaft cell on the ﬂy’s left side might sense more H than Su(H) and make a shaft (resulting in a normal bristle), while its counterpart on the right senses more Su(H) than H and makes a socket (yielding a “double-socket” phenotype). Because the same is true for H-deﬁciency heterozygotes, such asymmetries cannot be dismissed as being due to “leakiness” of alleles [767, 1998, 3067]. Rather, they are symptomatic of a breakdown in the “robust buffering” of the fate-assignment mechanism itself [1440, 3845, 4513].

22

IMAGINAL DISCS

Macrochaetes

a b

Key

SC PA NP PS HU VT PV OC OR DC SA P A P A P A P A P A D V P A P M A

4

5

100

H /+

3

% Present

s

1

0 100

w

H /H

2

w 0 100

w

H /H

s 0

d

c

basic

NH2

40

1059 a.a.

NLS (3) basic basic

COOH

*

**

Def./+

594 a.a.

Su(H)

+/+

COOH

* opa (2) * NLS (2)

e

20

100 a.a.

opa

Threshold Model

Dup./+

Su(H)

10

1

1 H

0

0 1

2 3 Doses of Su(H) +

4

Socket

Activity

Dup./Dup.

Activity

Number of macrochaetes in H s/+ flies

DNA-binding NH2

30

H

PRD

H Su(H)

0

Shaft

CHAPTER TWO. THE BRISTLE

Interestingly, shaft cells in H hypomorphs can display any morphology on the spectrum from shaft to socket (Fig. 2.6b) [198]. Likewise, overexpressing H during shaft morphogenesis (via a hs-promoter) can drastically deform the shaft: Unexpectedly, heat shocks after bristle determination affected morphogenesis of shaft and socket cells of macro- as well as microchaetae. At an early stage, very frequently shaft trifurcations were observed at the expense of the socket, the central shaft always being the longest. Later shocks caused a thickened shaft base with one to several spines. . . . If shocks were applied even later, shafts were completely absent, but instead, misshapen sockets were observed. This bald phenotype is inducible over a very long period until rather late stages of [the pupal period]. [2657]

Conversely, overexpressing N [3270] or Su(H) [3827] during that period partially transforms shafts into sockets, making them shorter and thicker. Intermediate shapes such as these are probably due to close contests where cells cannot select fates decisively. The intergradations also suggest that the “obvious” dissimilarity of shafts and sockets is deceptively superﬁcial. They may actually be outputs of the same dendrite-wrapping algorithm [1741, 2173]. If so, then shafts and sockets would actually differ quantitatively (e.g., in their extent of elongation). Little is known about the molecular machines that cells use

23

to modulate their shapes, and even less is known about the genetic circuitry that runs them (see below).

Several other genes help determine the 5 cell fates The aim of this chapter is to chart as straight a path as possible from Numb, which ordains cell fates from its perch at the cell membrane, to the genes that must implement those fates. Six players have been proﬁled (Numb, Delta, Notch, Su(H), Hairless, Tramtrack), a contender has been considered (Asense), and one putative set of implementing genes has been ruled out (the E(spl)-C). Below, this same route is retraced from surface to nucleus, with ﬁve more actors added, whose roles are less central: Serrate, Nak, Twins, Musashi, and Shibire. Until 1998, Dl was thought to be the only ligand for N in bristle development. This notion stemmed from the similarity of phenotypes in heat-sensitive Dl LOF and N LOF mutants. Both kinds of mutants exhibit the same “balding” (superﬁcial bristle loss) when exposed to pulses of high temperature around the time of SOP mitoses, and in both cases the 4-neuron clusters at many denuded sites indicate switching of IIa to IIb and sheath to neuron [1742, 3272]. However, when null alleles are made homozygous in somatic clones, Dl and N do not give the same phenotype: N null clones are bald, whereas Dl null clones make tufts of relatively normal bristles

FIGURE 2.6. Effects of Hairless mutations on bristle cell fates (a, b), and interactions between Hairless and Suppressor of Hairless

(c–e). a. Half head and thorax of a wild-type ﬂy (above), with all bristles omitted except macrochaetes. Beneath, macrochaetes (black circles) are seriated in posterior-to-anterior order, except that some are grouped [2560] as scutellars (SC), postalars (PA), dorsocentrals (DC), supra-alars (SA), notopleurals (NP), humerals (HU), verticals (VT), or orbitals (OR). Relative positions of intragroup members are posterior (P), anterior (A), dorsal (D), ventral (V), or middle (M). Presutural (PS), postvertical (PV), and ocellar bristles (OC; oval is an ocellus) are unpaired. The histograms show how each site is affected by Hairless genotypes. “H s ” and “H w ” are strong and weak LOF alleles of Hairless, respectively (“+” = wild-type). Black bars are frequencies (percent) of “normal” bristles (shaft and socket); shaded bars are frequencies of “double sockets,” which likely arise from a shaft-to-socket cell transformation (key at right). H function is evidently more limiting in shaft cells than in incipient SOPs because mild reductions in H levels (uppermost histogram) cause more double sockets than absent bristles [198]. The variation in sensitivity among sites makes no sense in terms of their seriation. b. Intermediate phenotypes between a normal bristle (type 1) and a “double socket” (type 5). In type 2, the shaft has abnormal ﬂuting and pigmentation, and the socket fails to form a complete circle around the base. In types 3 and 4, the shaft is a vestige that ultimately (in type 5) resembles a stunted socket. c. Number of “normal” (shaft and socket both present) macrochaetes on the head and thorax of H LOF /+ ﬂies (H 1 allele) carrying varying doses of the wild-type allele of Su(H). “Def.” and “Dup.” are a deﬁciency and duplication for the Su(H) locus. d. Domains in the H and Su(H) proteins (cf. App. 1). Dashed ovals connect regions needed for H-Su(H) binding [492]. “Basic” here means a segment of the protein where the average pI is 11 (sic!) [2657]. The DNA-binding domain of Su(H) is probably bipartite (i.e., same limits but centrally inert), given the properties of its mouse homolog [788]. e. Threshold Model of Bang et al. [197]. In this model, H and Su(H) titrate one another, and whichever protein remains undimerized determines cell fate. That is, excess Su(H) causes a cell to become a socket, and excess H causes it to become a shaft. N.B.: In other contexts, Hairless may act independently of Su(H) [725]. Data in a are from Table 2 of [198], b is a schematic of that article’s Fig. 5, c is replotted from [117], d incorporates data culled from [492, 1331, 2657, 3826], and e is based on [197].

24

(N.B.: In the original study [1797], Dl 9P39 patches made tufts, whereas Dl RevF10 patches – like N null – were bald, but a later study found no such allelic difference [4859], and both alleles may be nulls [3272].) In the absence of either N or Dl, all SOPs should have made neurons instead of bristles. How can normal bristles develop without Dl? The answer is a redundant ligand. Serrate (Ser) was known to encode a N ligand but was not suspected of acting in the SOP lineage because none of its mutant phenotypes (including null ones [4029]) involve bristle defects [2561]. However, Ser and Dl are related proteins [1252, 4302], and Ser (when artiﬁcially expressed) can substitute for Dl in embryonic neurogenesis [1634], implying an overlap in functional ability. Whereas Ser null clones look wild-type and Dlnull clones make tufts, doubly mutant Ser null Dl null clones are bald like N null and have extra neurons at denuded sites [4859]. Evidently, Dl and Ser both activate N in the SOP lineage, although Dl’s contribution is greater (and hence less dispensable) than Ser’s. (Why t.s. Dl LOF defects should be stronger than Dl null clonal defects remains unclear.) Consistent with this conclusion, shaft cells can be transformed into socket cells by overexpressing either Dl or Ser in neurons [2008] (Fig. 2.7c). A yeast two-hybrid screen [763, 1228] for Numb-binding proteins netted numb-associated kinase (nak), which encodes a putative serine/threonine kinase [765]. Nak interacts with Numb’s PTB domain, which is interesting from several standpoints: [4859].

1. Numb can localize properly without this domain [1307, 2269], so Nak probably does not escort or anchor Numb to the cortex [765]. 2. Like Nak, N binds Numb’s PTB domain [1307], so Nak might displace N from Numb, in which case it should antagonize Numb. Indeed, it does: overexpression of Nak alters cell fates in the opposite way from numb GOF [765]. 3. The PTB domain is a docking motif that binds phosphotyrosines [765, 2530], so Nak might hinder Numb indirectly by phosphorylating substrates that directly displace N. Nak’s true role will not be known until LOF alleles can be isolated, but the Numb-N mechanism must involve phosphorylation somehow because LOF mutations in twins can switch IIb to IIa just like numb LOF mutations [3904]. Twins is the regulatory subunit of serine/threonine phosphatase PP2A in D. melanogaster [4418]. Other twins LOF alleles interfere with mitosis [1535, 2761]. Such

IMAGINAL DISCS

effects are consistent with PP2A’s role in other species [830, 3352, 3904], and they suggest that Twins might be responsible for the cell-cycle dependence of Numb’s cortical localization [2267]. LOF mutations in musashi also produce numb LOF like phenotypes [3035]. This gene encodes an RNA-binding protein. Because Musashi protein is nuclear, it probably does not localize mRNA (like Staufen [478, 1321, 2526]) or regulate translation. It might control RNA processing (splicing? [193, 1100, 2186]) in a positive manner for numb, nak, twins, or Hairless, or in a negative manner for Dl, N, Su(H), or tramtrack. Indeed, Musashi has recently been found to bind and muzzle tramtrack transcripts ([3035] sequel). Ever since Numb’s cardinal role in dictating bristle cell fates was revealed in 1994 [3579], many pieces of the genetic puzzle have fallen into place. One remaining mystery [2268] is: What upstream cell-polarity cues cause Numb’s cortical asymmetry? During asymmetric mitoses, the Numb crescent always overlies one pole of the spindle [2267, 4028] . This coordination makes sense because otherwise the division plane could bisect the crescent and dispense equal amounts to both daughters [3580, 4875]. Embryonic neuroblasts (NBs) and ganglion mother cells also parcel Numb unequally. In their case, spindle orientation and Numb localization are both controlled by Inscuteable (Insc) and Miranda [4323] . Miranda is an adaptor protein that interacts with Insc and Numb [1322, 3893] . Numb’s N-terminal 227 a.a. sufﬁce for proper crescent formation in NBs [2269] and may be hooked to Insc via Miranda [3893]. However, insc LOF mutants do not show defects in bristles [3550] or larval sense organs [2327] (although IIb mitoses are misoriented [3630]), so Insc seems dispensable [2607]. Miranda is present in the IIb cell [3630] (despite an earlier report to the contrary [2680]), but its function there (if any) is unknown. Other links in the NB chain of command may also be irrelevant [2017], such as Bazooka (upstream of Insc [3803, 4709]) and “Partner of Numb” (downstream [2606]), which binds Numb and Miranda [2609]. This conclusion seems odd given the many similarities between SOPs and NBs [587, 1805] . For example, (1) IIb and IIIb divide along the apical-basal axis [1447, 3630] like NBs, which localize Insc apically [2328, 2607, 2609]), and (2) the homeodomain protein Prospero segregates basally in IIb and IIIb [1447], as in the embryonic CNS [477] , and helps decide cell fate in both systems [2680] . The current working hypothesis for Numb localization (in both SOPs and NBs) is that Numb is recruited to the cortex by the WD-repeat protein Lethal giant larvae and then driven to its ﬁnal destination by a polarized

CHAPTER TWO. THE BRISTLE

a

25

If input =

Numb and Notch

Dl Ser

Key:

Su(H)

or

and

Socket

H

Shaft x

b

0

1 and

1 1

or

1

1

1

IIa

or

0

0

1

neuron

0 or

1

1

1 1

0

0 1

Shaft

y

1

1

1

nt

1 and

u sh

0

1

Socket

and 1

1

1

nt

s

hu

x and z y

x 0 0 1 1

y 0 1 0 1

z 0 0 0 1

x y

x 0 0 1 1

y 0 1 0 1

z 0 1 1 1

or

z

intercellular

0 mutual antagonism

1 and

0

x y 0 1 1 0

1

0 or

x y 0 0 1 1

"driver" 1

1 and

1

0

mitosis

neuron

IIa

0

and 1

c

x

Socket

0 and

1 1

1

1

mitosis 1

y not

1 and

0

then output =

symbol

1

Socket

1 0

FIGURE 2.7. Circuitry that assigns shaft vs. socket cell fates in the SOP lineage. Logic symbols (key at right; cf. [2284, 4735]) denote

activation, inhibition, combinatorial criteria (“and” gate), and redundancy (“or” gate). The “not” symbol is a blend of genetic and electronic icons [1751, 2770], and the reciprocal “not” condition (“mutual antagonism,” “ﬂip-ﬂop,” or “seesaw”) means that if one partner is on (“1”), then the other must be off (“0”). These relations are summarized in the truth tables, where “1” means “true” and “0” means “false” [399, 1433]. The “driver” is the element that chieﬂy dictates the outcome, and “intercellular” connotes a ligand (arrowhead) and receptor (“V”). a. Core pathway of proteins that decide shaft vs. socket cell identities (cf. Fig. 2.2 and text for details). Abbreviations: Dl (Delta), H (Hairless), Ser (Serrate), Su(H) (Suppressor of Hairless). In plain English, this circuit means: “Activate the Notch receptor if it binds either Dl or Ser on an adjacent cell (but go no farther if Numb is present!), then let it bind Su(H) and (as long as there’s not too much Hairless around!) let the Notch-Su(H) complex turn on genes for socket identity; otherwise, turn on genes for shaft identity.” b. The circuit as it functions in daughters of the IIa cell (Fig. 2.1) in wild-type ﬂies. Numb is normally the decisive factor in setting cell fate (see text). The daughter that lacks Numb (above) has an active Notch pathway and hence forms a socket, while the daughter that inherits Numb (below) has an inactive Notch pathway and hence forms a shaft. c. The circuit as it malfunctions when neurons are forced (via neuron-speciﬁc Gal4-UAS driver “31-1”) to make excess Delta [2008]. In this situation, the prospective shaft cell makes a socket instead, presumably because hyperstimulation of Notch receptors supersaturates the limited amount of Numb. The ability of such excesses to “short circuit” the system (thick line) implies that care must be exercised in interpreting GOF phenotypes (in general) whenever proteins “ﬂood” a network.

26

IMAGINAL DISCS

cut

poxn

0 0 CT

1 1 CS

1. smell

1. stretch A CNS

A

A

N N

A

N CNS N 2. "fire"

N CNS

Ato

(Prepattern)

N

N

N

N 2. "fire"

Cut SOP: CT

and

and

SOP: CS GOF

Poxn

cut or

and

LOF

LOF

poxn

SOP: MS

GOF

1 0 MS

1. touch

3.

st

re

tc

h

ato

poxn

N

2. pivot

CNS

actomyosin motor [468,3205,3326], where it is anchored by Gαi (inhibitory subunit of GTP-binding protein) [3776]. Near Numb’s C terminus is a tripeptide – NPF (asparagine-proline-phenylalanine) – that binds “EH” (Eps15 Homology) domains [3725, 4417]. Because NPF or EH motifs are found in proteins that control clathrinmediated endocytosis [2699, 2756], Numb’s inhibition of N might entail effects on endocytosis. Indeed, Numb can inﬂuence the endocytic internalization of membrane receptors [3757]. Evidence that Notch signaling requires endocytosis comes from shibire (shi). Heat-sensitive shi LOF mu-

CNS

N 4. "fire"

tations cause the same anatomical defects as N LOF mutations and share the same sensitive periods [1057, 3423, 3425, 3863, 3891]. Among the defects are socket-to-shaft transformations [1803, 3425]. The protein encoded by shi is the ﬂy homolog of the mammalian GTPase “dynamin” [740, 4443]. In neurons, dynamin self-assembles around the necks of clathrin-coated pits, and a conformational change in these “collars” appears to pinch off vesicles from the membrane, although the mechanism may involve pushing instead of pinching [2795] . Detachment of vesicles halts in t.s. shi LOF ﬂies at high temperature [1016] , which explains why shi was recovered in a screen

CHAPTER TWO. THE BRISTLE

27

FIGURE 2.8. Circuitry (cf. Fig. 2.7 for key) controlling the identity of mechanosensory (MS) vs. chemosensory (CS) bristles vs.

chordotonal (CT) organs. Those organs are shown with their neurons (N) but without their support cells. They are created from single-cell SOPs (black rectangles) via differentiative mitoses (not shown; cf. Fig. 2.1). In the upper left of each panel are transcriptional states (1= on; 0 = off) of cut and poxn depicted as a binary code. The circuit (middle area) is simple. “Prepattern” genes (cf. Ch. 3) activate atonal (ato) at certain spots in the skin, and Ato suppresses Cut. If the SOP also lacks Paired box-neuro (Poxn), then it makes a CT organ. If Ato is absent, but other proneural proteins (not shown) are present (cf. Ch. 3), then Cut will be expressed and the cell will make a bristle. The bristle type will be CS if poxn is on and MS if poxn is off. The state of poxn (like ato and cut) must be set by region-speciﬁc genes upstream (not shown). Thus, LOF or GOF mutations in poxn interconvert MS and CS bristles, and cut LOF or ato GOF transforms MS bristles into CT organs. Transformations of bristles into olfactory sensilla (not shown) are elicited by amos GOF [1587]. CT organs lack protrusions [378, 1454, 2787, 2831] and arise from the SOP via a different lineage tree [1756]. Stretching of their dendrites (springs attached at two anchor points “A”) causes the neurons to ﬁre an action potential to the CNS [789, 2018]. CT sensilla on the ﬂy femur have 2 neurons [3867], whose cell bodies are embedded along the stretching axis (unlike depicted here). MS bristles transduce touch stimuli by lever action [2174]: deﬂection of the rigid shaft (triangle) toward the body surface (horizontal line) causes it to pivot. Neural depolarization appears to be due to stretching of the dendritic membrane (vs. compression of the tubular body) [789, 874, 2787] (but see [4527]). CS bristles should technically be termed “chemomechanosensory” because they use one neuron to sense touch like MS bristles, and they have four extra neurons whose dendrites extend up the shaft to a pore at the tip [2502, 4125], where they “taste” salts or sugars [111, 2955, 3061, 3529, 4841]. This schematic is adapted from [2174] (MS), [3529] (CS), and [378, 2128] (CT), with logic based on [1455]. Many authors have conjectured that cells differentiate by making a series of binary choices. A favorite metaphor for decision trees has been a rail yard of bifurcating railroad tracks [3063]. N.B.: Expression of cut at the wing margin goes through two phases; only the later phase controls SOP identity [353].

for conditional paralysis (viz., it stops synaptic vesicle recycling) [4759] . Shibire may also play other roles in vesicular trafﬁc [2232, 2900]. Endocytosis typically attenuates signaling by removing activated receptors from the surface [2031] and sending them to lysosomes for destruction [3217] . If true here, then blockage of endocytosis by shi LOF should enhance Notch signaling [702, 3863, 4482] but, as stated above, shi LOF actually causes a N LOF (not a N GOF ) phenotype. This paradox was ﬁnally solved in 2000 by using separate antibodies to follow the fates of N’s extracellular (N-extra) vs. intracellular (N-intra) domains [3271]. It turns out that N-intra cannot be released from the membrane of the “listener” cell until N-extra is engulfed by the “speaker” cell as a ligand-receptor complex. The engulﬁng process requires Shi [3271], and Notch signaling requires release [4156] – hence the similarity of shi LOF and N LOF phenotypes. Because Shi seems to not play a critical role in the listener cell, Numb is probably not acting via Shi in that cell. Notch may be the only ligand that needs endocytosis for transduction per se in discs because other ligands can signal without it [3863]. Nevertheless, endocytosis does appear to propagate certain signals within the epidermis (viz., Dpp [1169, 1546] and Wg [273, 2900, 2984]), perhaps via a “transcytosis” mechanism that uses separate sets of (nontransducing) receptors (cf. Ch. 5) [811, 1154, 2843, 4379, 4694].

Pox neuro and Cut specify bristle type The gene paired box-neuro (a.k.a. pox neuro or poxn) was isolated in a search for DNA-binding motifs of the “paired box” class [401] and was named “neuro” because it is transcribed in neural precursors of the CNS and PNS [950]. In imaginal discs, Poxn is expressed in SOPs of chemosensory (CS), but not mechanosensory (MS), bristles [149, 950]. CS bristles of ﬂies have many features that differ from MS bristles [4125]. For example, they have a shaft that is thin and curved (vs. thick and straight), a characteristic axonal CNS projection, behavioral reactivity to airborne chemicals, and a different lineage that produces 5 neurons (vs. 1) – 4 of whose dendrites extend to the bristle tip (Fig. 2.8) [1741, 1802, 3004, 3061, 3529]. On the dorsal tibia and wing margin, poxn null converts CS into MS bristles [149], while poxn overexpression has the opposite effect [149, 3141, 3142]. Evidently, the role of the wild-type poxn gene is to divert SOPs from a MS to a CS mode of differentiation. One aspect of CS bristles that fails to be transformed in poxn null mutants is the (earlier) times when SOPs arise [149], which may explain the larger bristle sizes (see below). Null mutations at the cut locus convert external sensilla into “chordotonal” (CT) organs in embryos [378, 2831], and a similar transformation (albeit often partial) is seen for adult bristles in cut-null areas of genetic mosaics. Overexpression of cut causes a reciprocal phenotype in embryos [372], although effects in adults have not been

28

reported. CT organs are stretch receptors that fasten to the cuticle (or muscle) at two points and normally lack an external projection (Fig. 2.8) [1454, 2787]. Aside from a sensory neuron (femoral sensilla have 2 [3867]) they have at least 2 support cells: a “cap” cell and a sheath cell whose “scolopale” abuts the neuron’s dendrite [378, 789, 2831]. In this regard, they resemble bristles, which may well have evolved from CT-like structures [378, 3437]. Chordotonal SOPs express atonal (ato) [4898], a bHLH gene located outside the AS-C (at 84F) [2040], but bristle SOPs do not (they express cut instead [149, 2000]). When bristle SOPs are forced to express ato, they stop expressing cut and transform into chordotonal organs [2038]. Evidently, the normal duty of ato is to ensure that SOPs develop into chordotonal organs by suppressing cut (ato cut). SOPs “know” whether to turn on ato due to cis-regulatory enhancers beside the ato transcription unit that respond to region- speciﬁc cues [4208]. Atonal also governs the development of photoreceptor cells (cf. Ch. 7). Based on the normal expression of poxn in cut null embryos [4481] and the normal expression of cut in poxn null discs [149], poxn and cut appear to act independently, despite initial reports (ubiquitous expression and reporter-gene studies) that suggested otherwise (cf. App. 4) [4481]. Apparently, SOPs “read” the state (on or off) of these genes and adopt a particular pathway of differentiation (MS vs. CS vs. CT) accordingly (Fig. 2.8). The failure of cut null to achieve a complete transformation implies that other genes may overlap cut functionally in setting organ-type identity. Candidates include the paired genes BarH1 and BarH2 [3763], which, like cut, are expressed in embryonic external sensilla [1843]. Mutations in these genes switch one type of sensillum to another: deletion of both genes converts “campaniform” to “trichoid” sensilla (see [2128, 2174, 2787] for normal anatomy and [2473, 4309] for transitional intermediates), and overexpression of BarH1 induces the reverse [1843]. Like cut, the Bar genes contain a homeobox [1842, 2286], which suggests that SOPs adopt fates via the same kind of logic that operates on a larger scale through bona ﬁde homeotic genes (cf. Ch. 8) [282]. Olfactory sensilla are governed by yet another bHLH gene named “amos” (absent solo-MD neurons and olfactory sensilla, where MD stands for multiple dendritic) [448, 1587, 1928]. Amos (at 36F) and Atonal (at 84F) are more closely related to each other than to the AS-C family (at 1B) [1756]. Indeed, their basic (DNA-binding)

IMAGINAL DISCS

domains are identical, except for a conservative Arg-toLys change. Nevertheless, they have dissimilar LOF and GOF phenotypes – presumably because different sets of cofactors bind idiosyncratic motifs outside their bHLH domains and steer them to different target genes [1587].

Bract cells are induced by bristle cells The bract is a noninnervated hair made by a single cell [3552, 4531] (Fig. 2.1). It differs from other “trichomes” [2876, 2877, 3362] insofar as it is thick, pigmented, and found only on legs and wings [1361, 1714, 4338]. On the legs, bracts reside next to MS bristles on segments distal to the trochanter [1883]. On the wing, they reside next to MS bristles on the proximal costa [524, 793]. Because the bract cell is not part of the SOP clone [544, 1356, 1800, 4334, 4344], it has long been thought to arise by induction [1808, 3421]. When disc cells are dissociated and reaggregated, isolated bracts are never seen [4334], implying a dependence on bristles. Indeed, bracts do not develop unless both the shaft and socket cells are present [1357, 1808, 3448]: 1. Injecting larvae with Mitomycin C [4335, 4531] or nitrogen mustard [4337] often suppresses bristle sockets, and bracts are missing wherever shafts lack sockets. Likewise, bracts fail to form when sockets are transformed into shafts by N LOF or shi LOF [1803, 3425]. 2. In the mutants Hairless 2 and shaven de (a LOF allele of dPax2 [1319]), bristle shafts are often absent or vestigial [2168], and bracts are missing wherever sockets lack shafts [4338]. Why do some bristles have bracts, while others – only a few cells away – do not? Bristles that normally lack bracts seem to be those that develop earliest (CS bristles and macrochaetes) [149, 1598, 1803, 3142, 3628]. Thus, it is possible that every SOP emits a signal at one stage of its differentiation, but if this stage fails to overlap the period when epidermal cells are responsive, then the bristle will lack a bract. Consistent with this conjecture, CS bristles can acquire bracts when t.s. N LOF or shi LOF mutants are heat-pulsed [1803] (because delayed signals now enter the competence window?), and MS bristles can lose bracts when wild-type pupae are heat-shocked [1803] (because signals are delayed beyond the end of the window?). The Notch pathway is also involved in allocating bracts to particular parts of the wing margin [793, 3092].

CHAPTER TWO. THE BRISTLE

Recruitment of cells by induction is also instrumental in the development of ommatidia in the eye (cf. Ch. 7), olfactory sensilla in the antenna [3531, 3548], and CT organs in the femur [4898]. The latter case is intriguing because it involves iteration (old SOPs inducing new ones) that could theoretically go on forever. In fact, the process is limited by the duration of the competence period and by the extent of the competent region [4898]. The CT inductive signal is transduced by the EGF receptor pathway [4898] (cf. Fig. 6.12). Evidently, this pathway also mediates bract induction because (1) Star LOF causes missing bracts, (2) Star LOF Ras1 LOF double mutants are missing even more bracts, (3) all bracts can be eliminated by heat-treating t.s. Egfr LOF mutants, and (4) ectopic bracts can be induced throughout the epidermis by heat-shocking hs-Ras1 GOF pupae (L. Held, unpub. obs.). Either the receptor (Egfr) or the ligand (unknown) must be localized in accord with proximaldistal cell polarity because bracts are only found on the side of the socket opposite the shaft, and this rule is obeyed even when bristles are misoriented in mutant phenotypes [1357, 1810]. Implementation of the received signal requires the homeobox gene Distal-less (Dll): (1) Dll is expressed in bract, but not bristle, cells on the femur [618]; (2) Dll LOF mutations suppress bracts [618, 4212]; and (3) Dll null clones lack bracts [618, 1561].

Macrochaetes and microchaetes differ in size but not in kind Unlike the sense organs interconverted by mutations in poxn, cut, BarH1, and BarH2, macrochaetes (MCs) and microchaetes (mCs) do not differ in kind. Regardless of size, all tactile bristles share the same cellular composition, lineage, and anatomy. Indeed, a full spectrum of bristle lengths is seen on the legs [1714, 1808, 1883], and mutations can cause notal bristles to have sizes between MCs and mCs [3067, 3373]. Thus, it is not surprising that no “selector” genes have been found that dictate MC vs. mC identity in the same way, for example, that poxn speciﬁes CS vs. MS bristles. Bristle size is a function of polytenization [2475, 4641] – a cyclin-dependent process of DNA replication [961, 1246, 1262, 2551, 4376, 4820] that enlarges the cell’s nucleus and cytoplasm without subsequent cell division [216, 1336, 1764, 3030, 3301] (cf. lepidopteran scales [2338, 4014]). In notal mCs (whose cells are initially diploid), the shaft cell undergoes two rounds of endoreplication, and the socket cell undergoes one [1741]. The degree of polyploidy achieved by MC shaft and socket cells is much greater [2475]. In

29

wild-type males, dorsocentral MCs average 336 µm (posterior) or 250 µm (anterior) in length, while nearby mCs are only ∼80 µm long [3067] and tarsal bristles are ∼50 µm long [1803, 2544]. When SOP mitoses end, the shaft cell is packed with rough endoplasmic reticulum as protein synthesis starts for shaft elongation and cuticle deposition [3216, 3552]. Indeed, ribosomes are probably working at top speed because reducing the supply of any one component slows the entire process. Thus, there are more than 50 sites in the genome where halving the gene dose (i.e., haploinsufﬁciency) causes the same short-and-thin “Minute” bristle phenotype [118, 2561], and the majority of these sites appear to encode ribosomal proteins [3718]. Likewise, bristle size depends on the number of ribosomal RNA genes (at bobbed loci on the X and Y) [1520, 1909, 3604, 4091, 4104]. The situation is analogous to the reduced wing size seen with LOF alleles of rudimentary: this “housekeeping” gene encodes a pyrimidine-synthesizing enzyme, which becomes rate-limiting during wing growth [1314, 1315, 1865, 3838]. Bristle length also depends on two bHLH-PAS genes – spineless and tango – but their role is unclear [1119, 1166]. Larger bristles typically have more longitudinal ridges (up to ∼10) [2474, 3362, 3832, 4639], probably because ridge interval is constrained by an invariant molecular feature such as the diameter of an actin ﬁlament bundle [1645, 1898, 3216, 3351, 3552]. The bundles (up to ∼20 around each shaft [94]) are organized by chickadee [4474], forked [3356], rotundRacGAP [1642], sanpodo [1128], singed [640, 3283], Stubble [94], and tricornered [1430] around the time of cuticle secretion [1642, 3552]. When seen from the side, the grooves in wild-type bristles seem to wind helically around the shaft [3583, 4096], but in fact they meet along a top seam [4639, 4641]. These chevrons suggest that bristles evolved from triangular scales, which rolled up into a cone [1340, 4640]. Shaft differentiation is enforced by dPax2 [1319]: LOF alleles remove shafts, and excess dPax2 elicits two shaftlike spikes from each socket cell [2168]. However, dPax2 probably operates more like a switch because dPax2 GOF can also convert neurons into glial cells [4303]. Bristle cells must divide, endoreplicate, and differentiate before cuticle deposition starts. The larger the bristle, the earlier it should have to begin these events in order to ﬁnish on time. This rationale explains why MC SOPs divide before mC SOPs [1741, 1925], and in general why bristle lengths are inversely correlated with the times of onset of differentiative events (cf. Fig. 3.4c) [806, 2837, 3420]. Similar reasoning explains why SOP mitoses

30

commence earlier for multiply innervated CS bristles than for singly innervated MS bristles within each body region [149, 1598, 1741, 1803, 3142]. They simply need more time to make more cells. If the number of replication endocycles is ratelimiting for bristle size, then delaying the time of SOP initiation should reduce the number of endocycles and cause a smaller bristle. Indeed, tiny bristles are often seen in ﬂies carrying subnormal doses of achaete or scute [801, 802, 4095, 4098, 4108], and the stunting may be due to late onset of differentiation [1462]. Conversely, prodding an SOP to arise prematurely should allow more endocycles and cause a larger bristle. MS bristles that develop from transformed CS SOPs in poxn null ﬂies are probably larger for this reason: the SOPs still arise at an early (unaltered) stage and hence have more time than neighboring MS SOPs to endoreplicate. SOP initiation can also apparently be accelerated by augmenting AS-C gene function [1563, 2690]: 1. Notal mCs can become MCs when lethal at scute is overexpressed (via a UAS-l’sc transgene driven by scabrous-Gal4) [3027, 3037]. 2. Derepression of the AS-C in the dorsocentral notum (in polychaetoid LOF mutants [733]; Ch. 3) changes mCs into MCs [3067], while the opposite transformation (MCs into mCs) is seen when spalt (an AS-C regulator) is overexpressed [987]. 3. When combined with mutations that derepress the AS-C, ac GOF (Hairy wing) alleles transform certain leg bristles into MCs [1802]. 4. Ectopic overexpression of sc in the eye can convert interommatidial mCs to MCs [4209]. 5. Extra doses of sc (in a background where sc is dere-

IMAGINAL DISCS

pressed) convert tergital mCs to MCs [2690], and similar enlargements occur when sc is overexpressed using a heat-shock promoter [3628]. (See [3905] for how wingless may be setting AS-C levels for this purpose.) When cells are forced to divide by artiﬁcially expressing the mitosis-licensing agent String, unusually small bristles often arise at sites where SOPs are being recruited [2225]. This effect has been attributed to a delay in SOP initiation (consistent with the above argument), rather than to precocious entry into the SOP differentiation pathway [2225]. In summary, it appears that bristle size is controlled by rheostats, rather than by switches. A bizarre bristle transformation occurs in ﬂies with a construct of the male-speciﬁc cDNA of doublesex (a gene involved in sex determination) driven by a heatshock promoter. Whereas most leg bristles in wild-type ﬂies are thin, tapered, and brown, most leg bristles in these ﬂies are thick, blunt, and black, like bristles of the sex comb [2106]. Whether this trait involves changes in the timing of SOP events remains to be determined. Evolutionarily, bristle development has been entrained by various anatomic gradients. For example, bristle lengths increase from anterior to posterior among notal MCs [805, 2837, 3067], tergital mCs [801, 2690], and sternital bristles [804]. They increase from dorsal to ventral for tergital MCs [801], from ventral to dorsal for tarsal bristles [1803], and from distal to proximal for leg bristles in general [1714, 1883]. Bristle intervals typically increase with bristle lengths. Various aspects of bristle patterns have also been tailored to ﬁt certain features of the cuticular landscape. Explanations for some of these correlations are presented in Chapter 3.

CHAPTER THREE

Bristle Patterns

The epidermis of a D. melanogaster adult has on the order of 500,000 cells, ∼5,000 of which (∼1%) make bristles [1804]. A priori, it would seem reasonable to expect bristles to sprout as randomly as the hairs on a human arm. However, even the most scattered bristles – the tergite microchaetes (mCs) – have fairly uniform spacing [801, 2301--2303]. At the other extreme of precision are the 40 macrochaetes (MCs) on the head and thorax, whose basic layout has been conserved for 50 million years (Fig. 3.1) [1625, 3966, 3980, 4185]. Except for the MCs, the bristles of each body region tend to vary in number and position from one ﬂy to the next. Interestingly, most bristles are organized in rows that run parallel or perpendicular to axes of the body or limbs [1808, 2883, 4015]. Within such rows, the bristles are aligned more or less accurately and are spaced more or less evenly. Different rules govern different patterns. Thus, notal mCs form jagged rows along the anteriorposterior axis [805, 4428], while wing bristles form straight rows along the margin [1741], eye bristles arise at alternating vertices of each ommatidium [3539], and belly (sternital) bristles are spaced at intervals proportional to their shaft lengths [804]. Why do such patterns exist? Surely, some are adaptive. For example, ﬂies use “brushes” (parallel transverse rows) on the legs to wipe dust from their eyes [4225], and other patterns appear to map air currents, prevent wetting, or act as shock absorbers [1116, 3206]. However, many may simply be accidents of evolution [3966]. Algorithms that evolved to align neuroblasts in the embryo’s central nervous system (CNS) may later have acquired the duty of building an adult peripheral nervous system (PNS) [3981, 3984]. Old tendencies to obey particular cues may

have been retained in the new disc-speciﬁc contexts [1804, 1807], causing bristles to sprout at certain spots, interfaces, or contour lines (cf. Chs. 5 and 6). Moreover, bristle patterns in different species may manifest whimsical idiosyncrasies. Variations within the genus [376, 1361, 1801] imply a frivolity that rivals the pastiches of colored scales on butterﬂy wings [210, 434, 3119]. Regardless of why bristle patterns arose, the designs themselves epitomize the general problem of pattern formation in development. Namely, what causes the correlation of particular cell types with particular positions? We are far from a complete explanation, but it is now clear that the diversity of the patterns is deceptive. In fact, all of them use the same genetic tools to enable each epidermal cell to make the Hamletian choice of whether to be, or not to be, a bristle. Given the two dimensionality of the cuticle and the single-cell origin of each bristle, the riddle posed by all these patterns is reducible to a simple Euclidean problem. To wit, what kind of geometry does the genome use to place points in a plane?” Throughout the twentieth century, many of the intellectual giants of genetics tried to solve this “Bristle Plotting Puzzle” by clever experimentation or deft theorizing. Most of the speculation focused on mutants with abnormal numbers of MCs. T. H. Morgan toyed with the genetics of extra-bristle mutants as early as 1915 [2640]. Morgan’s student, A. H. Sturtevant, was more intrigued with missing-bristle mutants, whose asymmetric patterns are as eye-catching as a gap-toothed smile. In 1918, he used Dichaete in artiﬁcial selection trials [4178]; in 1931, he toppled a popular theory about scute [4187]; and in 1970 (just before his death), he showed a dose

31

32

IMAGINAL DISCS

Lattice Tandem Regular Row Irregular Row Constellation Isotropic

FIGURE 3.1. Diversity of bristle patterns on the ﬂy surface.

In this adult male, the eye bristles occupy alternating vertices of ommatidia in a hexagonal lattice. The sex comb typiﬁes a “tandem” array where bristles touch. Other leg bristles are aligned in straight (“regular”) rows and spaced evenly. On the notum the rows tend to be irregular, especially laterally. The four scutellar macrochaetes form an aperiodic (“constellation”) pattern that is constant from one ﬂy to the next, as are the arrangements of other macrochaetes. Most tergite bristles (except those at the posterior edges) are arranged randomly (“isotropically”) but spaced uniformly. Tergite pigmentation has been omitted for clarity. Adults are ∼3 mm long [3421]. Adapted from [1804]. For anatomical nomenclature, see [1224]. See also App. 7. What sort of geometry does the ﬂy use to create these patterns? Do all the arrays arise via the same algorithm? Do bristles know where they are? The mysteries of this “golden ﬂeece” have intrigued investigators for decades [1799, 1804, 1808, 2434, 4096].

dependency of achaete and hairy that later proved prescient [4185]. Another famous member of the Morgan lab, H. J. Muller, inspired a team of Russian researchers to dissect the achaete-scute Complex (AS-C) [650] – an enterprise that later became a cottage industry for Antonio Garc´ıa-Bellido and his school in Madrid [1354, 1452]. British patriarchs John Maynard Smith [2768, 2769] and C. H. Waddington [4517] invented cute schemes for categorizing mutant patterns, while Vincent Wigglesworth and Peter Lawrence speculated about the chaotic origins of natural patterns [2427, 2439, 4657]. Lawrence and Garc´ıa-Bellido used bristle patterns as a platform for

digging into deeper issues of development, and these two mentors trained many talented students who became leaders in this ﬁeld. Indeed, bristle pattern research is so interesting from a historical standpoint because of these crosscurrents. Its parochial models have often inﬂuenced the larger ﬁelds of development and genetics and vice versa. For instance, Curt Stern’s ideas about bristle patterning (discussed below) colored Lewis Wolpert’s thinking about patterning in general [4724], and Wolpert’s theory of positional information in turn revolutionized the bristle subﬁeld via the team founded by Howard Schneiderman

CHAPTER THREE. BRISTLE PATTERNS

and Peter Bryant in California [530, 1485]. Most of the old questions have now been answered by modern molecular techniques, but the story of how we got here is as charming as the answers themselves.

Surprisingly, different macrochaete sites use different signals Alan Turing, the British mathematician who cracked Germany’s Enigma Code in World War II [1863], is also famous for helping to found the modern ﬁeld of computer science [872, 4409]. It is less widely known that Turing also dabbled in developmental biology. In 1952, he proposed a new theory to explain how biological patterns arise [4410]. He argued that chemical reactions among randomly diffusing substrates should cause the products to accumulate at regular intervals. Given the right parameters, his formulae predict stable spots or stripes whose spacing depends on diffusion constants, and whose arrangement depends on the shape of the region where the reactions occur [1176, 2769, 3014]. Turing’s ideas founded a ﬂourishing school of thought about the abilities of reaction-diffusion systems to create patterns [1727, 2806, 3013]. In 1954, Curt Stern invoked comparable dynamic forces to explain how bristle patterns develop [4095, 4096]. Stern (1902–1981) had learned ﬂy lore as a postdoc in Morgan’s lab [2614, 3070], where he earned kudos for proving that crossing over occurs in somatic tissues [119, 4089, 4347]. Poetically, this very phenomenon allowed him to test his new ideas [4100, 4103]. He acknowledged that chemical interactions such as those in Turing’s Model could mediate patterning, but he thought that physical forces might work just as well. For example, Stern conjectured that uneven growth in a sheet of cells might lead to stress points, which cells could then “read” as signals for making bristles. The initiation of differentiation of specifically localized organs cannot be a purely locally caused process but is possible only on the basis of a prepattern which itself is the result of interrelation of parts of a developing whole. [4098] Possibly bristles differentiate at places of specific strains or stresses set up in the folded embryonic tissue which later smoothly covers the thorax. . . . Of course, the physical scheme of tensions used in this train of thought is only one of many variants. Different general patterns of biochemical properties . . . in the embryonic future epidermis could be considered equally well. [4096]

Stern coined the term “prepattern” for the array of signals that preﬁgures the ﬁnal pattern. Initially, he imagined that the sites containing the cues must be in-

33

terdependent. He called those sites “singularities.” In the above example, the singularities were the stress points, but in general they could be any heterogeneity. This idea came to be known as the “Prepattern Hypothesis” [4346]. In a population of somatic cells which are genetically alike, the differential fate of some specially located ones must be due to a superimposed differential organization of the population. . . . The existence of a differential organization, a ‘‘prepattern,’’ within any imaginal disc must precede and hence be responsible for the patterned origin of bristles. [4095]

Because the prepattern was deemed to be a gestalt, any change in one of its components should alter the remaining components in the same way that tugging one node of a spider’s web will alter the whole web. To test whether inﬂuences spread beyond the point of an interference, Stern used mosaic ﬂies with discrete regions of mutant and wild-type tissue. He created the genetic patchiness by somatic crossing over. His reasoning was straightforward. If a mutant ﬂy differs from the wild-type in its network of force vectors, then mutant tissue in a wild-type background should cause novel webs of interactions, and strange new patterns should emerge that might yield clues to the mechanism. Over the next ∼20 years, Stern and his co-workers tested this “Force Field Model” by analyzing ∼20 possible “prepattern” genes in mosaic settings. Disappointingly, they found virtually no new patterns [4100, 4346]. Nevertheless, some clues were uncovered that led to profound insights. Ironically, the pithiest clues came from one of the ﬁrst mutations that Stern studied – ac1 , an allele of achaete (ac), whose name means “no bristle” in Latin. This gene was mentioned earlier as part of the AS-C (Fig. 2.4). Homozygous ac1 ﬂies usually lack a notal MC called the “posterior dorsocentral” (PDC). In mosaics, this PDC bristle is typically absent if its site is occupied by ac1 tissue and typically present if heterozygous tissue resides there [4095, 4096]. Evidently, cells at the PDC site use their own genotype to decide whether to make a bristle, regardless of the distribution of genotypes elsewhere in the disc. This local autonomy was not what Stern expected based on his Force Field Model, nor was it what Turing’s Model would have predicted since diffusing chemicals should naturally affect nearby cells [2769, 4016]. Given the ability of ac1 tissue to suppress the PDC bristle but virtually no other MCs when located at extraneous sites, Stern surmised [4100] that

34

IMAGINAL DISCS

Prepattern

Pattern

Force Field Model Filter Model Prepattern A

Competence Signals

Receptors A B C D E F

B

C D E H

H I J K L

F G J

I Filter

K L

Bristle Differentiation

CHAPTER THREE. BRISTLE PATTERNS

1. ac1 must interfere with the “competence” of cells to respond to a speciﬁc signal at the PDC site. 2. Different MCs must use different signals. His second deduction is a special case of the general Principle of Nonequivalence [2516, 4727, 4729] – the idea that identical structures are speciﬁed by nonidentical signals. This notion is counterintuitive because a priori Occam’s Razor suggests a minimum number of signals for any given pattern. In a similar way, stripes of pair-rule gene expression in the embryo blastoderm were once thought to arise via spatially periodic signals in a Turinglike process [1728, 2159, 2377, 2379, 3031]. However, there, too, the Principle of Nonequivalence trumped Occam’s Razor: most stripes are actually governed by unique combinations of transcription factors [104, 1908, 3247, 3587, 4834]. Thus, MCs – like pair-rule stripes – are made “inelegantly” [49, 2378, 2765, 3248]. Based on the above conclusions, Stern revised his model by inserting a new step (Fig. 3.2). For a prepattern signal to evoke a pattern element in the ﬁnal array, this signal must ﬁrst be transduced by the cell at that site. To illustrate this “competence” step, he used a prospecting analogy where signals differ qualitatively from one another: This then is a picture of gene action in differentiation. A whole developing embryo, or a developing part of an embryo, possesses regional differences of varied nature. One gene (or its products) responds to one specific regional peculiarity, another gene responds to other regional characteristics. These responses, which are the necessary prerequisites for a complete series of processes, result in specific differentiations. One may compare the groups of genes present in all regions

35

of the developing organism to groups of prospectors working in different regions of a country. Each group would be alike in consisting of different men looking for coal, gold, uranium, and so on. In some regions only the coal prospectors would respond to the coal deposits, at other places the gold prospectors would respond to gold veins, at still other places the uranium prospectors to ores of this metal. Each response would depend on the pre-pattern of geological differentiation. Each response would be the necessary prerequisite for subsequent differentiation in the form of the specific structures erected by mining engineers -- perhaps deep shafts and elevators for coal mines, surface excavations and washing troughs for gold, shallow tunnels and radiosensitive installations for uranium. [4096]

In this new formulation, mutant genes that affect the pattern should be classiﬁable into those that do so by altering the prepattern (“prepattern genes”) and those that do so by modifying cellular competence (“competence genes”). Signals that cannot be transduced due to a competence mutation like ac1 would be “ﬁltered out” before they elicit bristles at those sites [4100]. One way to envision this “Filter Model” is with cell surface receptors serving as the ﬁlter (Fig. 3.2), although Stern never advocated any particular molecular device. If each bristle site has a unique signal and each signal has a unique receptor, then the failure of ac1 cells to make a PDC bristle could be due to a defective receptor. In fact, the ac gene does not encode a surface receptor, nor does the ac1 mutation disable the Ac protein. Rather, ac encodes a nuclear protein, and ac1 deletes a region-speciﬁc cis-enhancer that lies 5 kb upstream from the ac gene’s transcription start site. As explained below, it is the ensemble of these enhancers at the AS-C locus that actually “ﬁlters” the prepattern signals.

FIGURE 3.2. Curt Stern’s Prepattern Hypothesis in its original (above) and ﬁnal (below) forms, both of which were proposed in

1954 [4095, 4096]. Force Field Model. Initially, Stern envisioned patterns as the steady-state outcomes of dynamic physical or chemical forces. In this illustration, uneven growth deforms the epithelium so that each half mesonotum acquires 7 stress lines. Each of the 11 sites (“singularities”) where the lines intersect then induces a MC. Because mutations can only change the pattern by altering the force vectors, any patches of mutant tissue in a wild-type background should distort the entire web of forces. Contrary to expectation, virtually every pattern-altering mutation that Stern studied (including achaete1, Fig. 3.3c) affected differentiation autonomously in mosaics. Filter Model. To explain why cells exhibit such insularity, Stern postulated that (1) each MC must use a different signal (A–L, although signals could differ quantitatively instead), and (2) a MC will only form at a site if the cell there is “competent” to respond to that speciﬁc signal. Inserted between the prepattern and the pattern is an extra step wherein each cell “ﬁlters out” signals that it cannot “hear.” In this illustration, the competence of each cell (depicted with apical microvilli) depends on surface receptors. In reality, positional signals are “ﬁltered” through enhancer elements within the achaete-scute Complex (Fig. 3.4) [2891]. Wild-type cells (like this one) cannot respond to Signal G, a “cryptic singularity” that Stern discovered [4097]. Similarly, ac1 cells would be “deaf” to Signal I (not shown). N.B.: Stern described these variations of his theme in general terms, but never formalized or named them as speciﬁc models. Here they are made explicit to highlight their differences. The idea that tensions affect gene expression (top model) seemed farfetched at the time, but various examples are now known [301, 761, 762, 1942, 2580].

36

Prepatterns may contain hidden ‘‘singularities’’ In male ﬂies, the foreleg bears a “sex comb” [1716], socalled because its single ﬁle of stout bristles resembles a hair comb [4344]. In gynandromorphs, sex comb “teeth” can arise when even a small patch of male tissue (in an otherwise female leg) overlaps this site [3441, 4105]. Evidently, female forelegs have inductive signals for teeth but lack a comb because female cells are not attuned to them. This case is interesting because it shows that even wild-type cells routinely “ﬁlter out” certain signals in their own epidermis. Extending this reasoning into the realm of evolution, Stern argued that signals may arise evolutionarily before the ability of cells to respond to them: Many Drosophila species are known which do not possess a sex comb in either sex. D. melanogaster undoubtedly evolved from such sex-combless ancestors. Must we postulate that a basically new configuration of a foreleg had to originate in evolution before a sex comb made its appearance; or can we not more simply assume that the sex comb pre-pattern was long in existence in the legs of flies and had to wait only for a mutation toward a single new gene which could respond to the hitherto unheard call? [4096]

In other words, ancestral drosophilids may have possessed a signal for sex combs, but this signal failed to evoke comb development until D. melanogaster males evolved the ability to “hear” it. Stern called such hidden signals “cryptic singularities” [4100], and he uncovered another instance in studying a certain duplication of the yellow gene, where AS-C enhancers reside. D. melanogaster normally lack a MC at the “interalar” site [3966, 4185] (“G” in Fig. 3.2) on the notum (dorsal thorax). However, an extra MC can arise there when the site is occupied by duplication-bearing cells, even if the rest of the notum is wild-type. Apparently, the duplication enables cells to “read” a subliminal signal that wild-type cells cannot [4097]. Because other (ancestral?) dipterans have an interalar bristle, D. melanogaster may have retained a vestige of the signal but lost the ability to transduce it, in which case any mutation that reveals it would be considered atavistic [1354]. Other authors have adduced evidence for cryptic singularities in line with the dorsocentrals [2640, 4507], between the ocellars and orbitals [2769, 4013], between the anterior and posterior scutellars [1282, 2413, 3890, 4517], and on the tarsus [1802]. Indeed, subthreshold bristle-forming tendencies have been found in nests of cells that nor-

IMAGINAL DISCS

mally do not form a bristle, but can be prodded to do so in certain mutant backgrounds [589]. There are two ways of thinking about singularities in general, regardless of whether they are overt or covert: 1. Singularities are instructive signals, tantamount to telling a cell to “Make a bristle!” 2. Singularities are not signals per se, nor are they inherently tied to any particular structure. Stern favored the second view [4098], as illustrated by the following passage where he cites some classic experiments of Spemann’s [4030]. There are some famous experiments in which embryonic tissues of a salamander have been transplanted into the mouth region of frog embryos, and reversely frog tissue transplanted into the mouth region of salamander embryos. These experiments have shown that both salamander and frog are endowed with a prepattern which distinguishes their mouth regions from the rest of the body. . . . In the evolution of the two branches of amphibians to which salamanders and frogs belong, no more than the appearance of new genes which responded in different ways to the previously established inductive capacity of the mouth region was required. The complexities of any organism must imply an inexhaustible array of prepatterns, a few of which are realized in accomplished differentiations and most of which are waiting for the response of genes not yet arisen in a specific organism. [4096]

The molecular-genetic analyses described below support this argument. Singularities appear to be transacting factors that elicit bristles only because the AS-C has cis-enhancers to bind them. In principle, any bristleless site could be a “cryptic singularity,” because evolution could endow the AS-C with enhancers to “read” virtually any position-speciﬁc protein (cf. Ch. 8) [662]. Thus, each prepattern is an accidental result of the cross-linking (by evolution) of bristle differentiation to certain heterogeneities of gene expression in the epidermis. Our understanding of prepattern mechanisms rests mainly on studies in the wing disc, where many transacting factors are known. Those agents will be examined in Ch. 6 after readers have been briefed on the key signaling pathways (cf. Table 6.2 and Fig. 6.14). The rest of this chapter focuses on the AS-C itself and its regulation by circuits that operate wherever bristles arise.

How Achaete and Scute control bristles was debated for decades Among ac1 mosaics that lack a PDC due to homozygous ac1 tissue occupying that site, a MC often arises

CHAPTER THREE. BRISTLE PATTERNS

nearby in heterozygous territory (Fig. 3.3) near the normal site [802, 3613, 4095, 4098]. This apparent ability of the PDC to be displaced led Stern to infer that the PDC can come from any cell within a cluster of equivalent cells at this spot. When the cell at the “preferred” site within this “equivalence group” is prevented from becoming a MC, another cell can substitute. Why don’t all of these cells make MCs? Stern guessed that whichever cell emerges as the SOP must block the others from adopting this fate. He likened the situation to embryonic ﬁelds, where the tissue area that can potentially make a structure (e.g., a limb [1955, 4589]) is typically larger than the region that actually does so. This astonishing result fits perfectly well into existing concepts of the embryologist. He has discovered the existence of pre-patterns which he calls embryonic fields. These are areas in which a specific differentiation may occur anywhere. Actually, under normal circumstances, the differentiation takes place in only a limited part of the whole field, at a peak, figuratively speaking. Once differentiation has set in at the peak, no other differentiation occurs within the larger field. If, however, differentiation at the peak is suppressed, then a lower region of the field may differentiate. This differentiation itself will exclude other differentiation within the remainder of the field. . . . The pre-patterns of the embryonic tissue of Drosophila, which call forth the response of genes involving the differentiation of bristles, are embryonic fields of larger dimensions than the limited points of normal location of bristles. If the influence of the achaete gene at the normal point prevents differentiation of a bristle, other parts of the field may assume the properties of peaks and differentiate bristles. [4096]

Given the directionality of the displacements, Stern deduced that the PDC cluster must be elongated along the anterior–posterior axis, rather than having the circular shape expected if the sensory organ precursor (SOP) emits a diffusible inhibitor.

37

tant ﬂies with null alleles for both genes. Such ﬂies lack virtually all bristles due to a failure of SOP inception [912] but otherwise look normal [1379, 1462, 1802, 3812]. Not only are ac and sc necessary, they are also sufﬁcient for bristle initiation because overexpression of either gene can induce extra bristles at ectopic sites [191, 635, 1348, 3628] by evoking extra SOPs [912, 3982]. AS-C polymorphisms account for much of the naturally occurring variation in bristle number seen in this species [2593, 2645]. Historically, this gene complex was viewed as holding the key to deciphering bristle patterning [1354, 4766], and rightly so. Partial-LOF sc mutants are striking because they create holes in an otherwise perfect pattern of MCs, and because the bristles that they remove depend strictly on the allele. Among the 11 notal MCs {PSC, ASC, PPA, APA, PDC, ADC, PSA, ASA, PNP, ANP, PS} (Fig. 3.4), the subsets that tend to be missing include sc1 {PSC, ASC}, sc4 {PSC, ASC, PPA, APA, ASA, ANP, PS}, sc6 {ANP}, sc7 {PSC, ASC, PPA, APA}, and sc9 {PSC, ASC, ANP} [1360]. How does the AS-C encode bristle positions? This “Bristle Coding Enigma” bafﬂed geneticists from the discovery of the ﬁrst sc LOF allele (sc1 ) by Calvin Bridges in 1916 [470] until 1995 when the mystery was solved by Juan Modolell’s team in Madrid [1538, 2890]. The intervening 80 years were punctuated by intriguing clues and colorful controversies [628, 650, 1354, 1799, 2891]. Some milestones in this saga are recounted below. 1916

It seems that differentiation of a typically located [PDC] inhibits other nearby potential bristle differentiations in the line defined by the position of the dorsocentral bristles but that absence of the [PDC] permits the realization of some of these potencies. [4095]

The inhibitory mechanism is considered later. It does not appear to depend on achaete or any other genes in the AS-C. The AS-C spans ∼100 kb near the tip of the X chromosome (Fig. 3.4). Four of its genes – achaete, scute, lethal-at-scute, asense – are termed “proneural” because they promote neural development in the CNS or PNS [1454, 3637]. Innervated bristles depend almost exclusively on achaete (ac) and scute (sc), as shown by doubly mu-

1920s

On January 22, the ﬁrst scute mutant was found by Calvin Bridges [470]. Twelve days later (Feb. 3), the ﬁrst achaete mutant was found by Alexander Weinstein. Both men were then graduate students in Morgan’s lab [2283], where the Great Quest for mutants had begun in 1910. Neither achaete nor scute could be given a one-letter abbreviation because “a” and “s” had been taken by arc and sable (in 1912) [470]. Hence, the two genes became known as ac and sc. Following H. J. Muller’s ﬁrst sojourn in the Soviet Union, A. S. Serebrovsky used Muller’s X-ray method to induce new sc mutations [650, 651]. His assistant, N. P. Dubinin, found partial complementation among alleles [650] and used these data to deduce a linear array of sc “subgenes.” Each subgene was supposed to control a discrete subset of bristles [1108, 1109]. His inferred sequence of subgenes

38

IMAGINAL DISCS

wild-type

achaete1 (y null)

mosaics

PDC

a

b

Proneural Cluster

c

e

d

f

Proneural Cluster (Remnant)

Proneural Cluster Halo PDC

g

1931

h agrees roughly with what we now call “enhancers” along the AS-C [1360, 1453]. A similar mistake was made at the Bithorax Complex [2507, 2782], where enhancers were thought to be genes per se before DNA cloning clariﬁed the situation [146, 1121, 2672, 4671]. A. H. Sturtevant and Jack Schultz supported the “linear array” tenet of Dubinin’s “Sub-

ADC

ADC

PDC

PDC

i

j gene Hypothesis” but disproved the “discrete subset” tenet by showing that allelespeciﬁc bristle groups can be modiﬁed via changes in the genetic background [4187]. This malleability implied that the patterning of bristles is “a system in which limiting factors are important and in which, therefore, changes in the rest of the system make

CHAPTER THREE. BRISTLE PATTERNS

39

FIGURE 3.3. Proneural clusters, whose existence was deduced by Stern in 1954

[4095]

and conﬁrmed histologically in 1989–91

[912, 3637, 3982].

a–f. Stern’s reasoning, based on the nonautonomous formation of an ectopic MC by wild-type tissue in achaete1 (ac1 ) mosaics. a. Heminotum (minus humerus) of a wild-type ﬂy, with all bristles omitted except the PDC MC. b. Homozygous ac1 ﬂies typically lack a PDC. Absence of shading indicates yellow null (ynull , yellow cuticle), which was a linked marker for ac1 1 1 null ac1 sn1 /+++ [636, 1442], as was singed (sn , gnarled bristles). c, d. Gynandromorphs arising from X-chromosome loss in y heterozygotes (redrawn from [4095]). Both heminota lack a PDC at its normal site, evidently because the tissue there is mutant. In some cases (e.g., d), a PDC-like MC arises in nearby heterozygous tissue [3613, 4095, 4096]. e, f. Illustrations based on Stern’s hypothesis for bristle displacement [4095]. Squares show imaginary details for PDC areas in a and d. Dashed line marks the normal PDC site. A “proneural cluster” (dark shading) of cells (small hexagons) is “competent” to make a PDC. The central cell becomes the PDC (e) unless its ac1 genotype renders it unable to do so (f), whereupon a cell in the remaining part of the cluster substitutes, thus displacing the PDC. All SOPs, Stern argued, must inhibit their neighbors from making bristles. g–j. Drawing of a real cluster on a right heminotum at ∼25, 20, 10, and 0 h before pupariation, as revealed by antibodies to Scute [912, 3982]. Ovals are nuclei (diameters ≈ 4 µm). The actual dorsocentral cluster (1) has an irregular shape, (2) grows, and (3) yields two MCs whose SOPs arise at the cluster’s edge. g. Some cells express more Scute (dark shading). h. The SOP for the PDC (black) arises in a high-Scute area and acquires a “halo” of low-Scute cells. i. As the cluster grows, the high-Scute area shifts and the ADC (no halo yet) arises. The reason for this shift is not known. j. In this cluster, non-SOP cells stop expressing Scute before SOPs, which do so later (before dividing). Further growth widens the gap between the ADC and PDC. As for why the ADC and PDC typically form on the medial (dorsal) side of their common cluster, the answer appears to be that a diffusible bristle-promoting signal (Dpp) encounters this cluster on its medial face and then wanes (cf. Fig. 6.14) [3373, 4368].

1932

1935

changes in threshold values.” Dosage thresholds have since been shown to be decisive in bristle patterning and to depend on a balance between opposing groups of HLH genes (see below). Sturtevant and Schultz advocated a version of Plunkett’s “Spreading Model” [3405], wherein a dorsocentral source (“center”) emits a bristle-inducing substance, and different sc alleles impede its diffusion in different directions by acting at different times. In the same year, Richard Goldschmidt (Curt Stern’s colorful mentor [4102]) proposed a similar diffusion-based theory [1520]. Muller [2986] and Sturtevant [4181] independently disproved the Spreading Model by demonstrating the same autonomy for sc that Stern later showed for ac [4095, 4096]. To wit, they showed that scLOF tissue can remove bristles without affecting nearby wildtype tissue – a result that is inconsistent with the involvement of diffusible factors. Our modern view of AS-C function afﬁrms Muller’s conclusion that “the development of bristles . . . is not governed by one or a few centers, but is in its major features autonomous at the site of each bristle.” Like Sturtevant and Schultz, George Child disproved Dubinin’s “one-subgene-onebristle-group” idea but did so by manipulat-

1954

1975

1978

ing the environment instead of the genetic background. He showed that allele-speciﬁc traits can be changed by raising scLOF ﬂies at different temperatures [767--769]. Curt Stern proposed his Prepattern Hypothesis [4095, 4096]. The autonomy that he found in ac mosaics ruled out a Force Field Model for prepatterns. He concluded that cells respond to position-speciﬁc, bristle-inducing signals only if they are “competent” to receive them. Displacements of wild-type bristles led him to postulate that (1) each MC arises within a group of equipotent cells, any one of which can become a bristle; and (2) the nascent SOP inhibits its neighbors from adopting a bristle fate. Clifton Poodry used irradiation to probe the timing of bristle differentiation on the notum [3420]. He found that when MCs are grouped according to the time when they acquire insensitivity to irradiation, the groups do not match the subsets removed by scLOF alleles. Thus, those subsets cannot be explained by loss of sc function at allele-speciﬁc times (as Sturtevant and Schultz had imagined). The time when a bristle site becomes insensitive to irradiation was found to be correlated with bristle length [2837]. This trend is probably due to a need for larger bristles to begin differentiating earlier in order to

40

IMAGINAL DISCS

Macrochaetes

a SC PA DC SA NP PS HU VT PV OC OR P A P A P A P A P A D V P A P M A

70 kb

60

y

82

50

40

20

10

0

l’sc sc AS-C

ac

64

30

-10

-30 kb

-20

ase T1

T2

ac1

b SC PA DC SA NP PS HU VT PV OC OR P A P A P A P A P A D V P A P M A

0

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15

Onset of radio-insensitivity (h AP)

Bristle length (arb. units)

c

80 pSC aPA 70 pDC

aSC

60 pSA 50

aNP aVT OC

40

aDC dHU

PS pPA aOR PV

30 20

0

1

2

3

4

5

6

7

8

9

10 11 12 13 14

Onset of radio-insensitivity (h AP)

1985

ﬁnish before cuticle deposition. This discovery (Fig. 3.4c) had no bearing on why different scLOF alleles affect speciﬁc bristles. Cloning of the AS-C was reported by Juan Modolell’s team [636], which had previously

cloned the sc portion [656]. Most sc mutations were found to map within a 50-kb region of DNA proximal to the sc gene. Nucleotide sequencing revealed similarities (and redundancies) of the four bHLH genes in the

CHAPTER THREE. BRISTLE PATTERNS

41

FIGURE 3.4. Spatiotemporal control of macrochaete development on the head and thorax.

a. The genotype-phenotype “switchboard” for MCs. Half head and thorax of a wild-type ﬂy (dorsal view; mCs omitted). Black circles below symbolize MCs seriated in posterior-to-anterior order, except that some are grouped (cf. Fig. 2.6 for key: P = posterior, A = anterior, D = dorsal, V = ventral, M = middle). The double helix spans the ∼100-kb achaete-scute Complex (AS-C) near the tip of the X chromosome. Arrows denote transcription units (kink = intron), with distal to the left and zero as an arbitrary EcoR1 site. Four “proneural” genes (achaete, ac; scute, sc; lethal-at-scute, l’sc; asense, ase) encode bHLH proteins, although only ac and sc normally affect MCs. Enhancers (rectangles) were mapped genetically (light shading; double arrows mark unknown limits) [636, 3688] or by their ability to drive reporter genes in proneural clusters (dark shading) [1538, 2891]. The minimal effective sequence per rectangle is unknown except for the DC enhancer [1380]. Note that the MCs and enhancers are not colinear (dotted lines). Genetic loci for ANP and APA enhancers (5–7 kb and −1 to −7 kb, respectively; not shown) differ trivially (a few kb) from those depicted (as assessed by reporters). The ac1 allele affects DCs (Fig. 3.3) due to an 18-kb (64–82) deletion that overlaps the DC enhancer. The functional core of this 5.7-kb enhancer is really only ≤1.4 kb long [1380]. The DC enhancer may be looped into contact with the sc promoter (30 kb away) when Pannier (a transcription factor) binds this enhancer and forms a complex with bHLH dimers that bind the promoter [3504]. The yellow gene (y) has its own enhancers (not shown) [1442, 1534]. In Ref. [1538], ASA and PPA are (mistakenly?) ascribed to the same enhancer. See also App. 7. b. Times (hours after pupariation) when sites become insensitive to γ-irradiation. Because the high dose that was used (104 Rad) kills dividing cells but spares postmitotic ones, onset of insensitivity probably signiﬁes completion of SOP mitoses [3420]. Indeed, onset times agree roughly with histological detection of four SOP progeny at these various sites [1925]. More precisely, times may reﬂect the start of endoreplication because shaft cells become insensitive and endoreplicate ∼1–3 h before socket cells [1741, 3420, 3421]. No colinearity exists between onset times and the overall posterior-anterior seriation (dotted lines), although posterior bristles precede anterior ones within most pairs – a correlation that may have less to do with location than with shaft length (see c). Times for mCs are 19–24 h AP (not plotted). c. Inverse correlation between bristle length and onset of radio-insensitivity. This trend may stem from a need for SOPs to begin mitoses or endoreplication early enough to ﬁnish differentiating before cuticle deposition. Strangely, the earlier a MC SOP arises (cf. Fig. 3.7), the longer it waits before starting mitosis [1925]. Lengths (arbitrary units) are for wild-type ﬂies raised at 17◦ C [2837], times are from [3420], and the line was plotted by best-ﬁt regression. N.B.: For calibration purposes, the actual lengths for pDC and aDC bristles are 336 µm and 250 µm, respectively [2544].

1987

1989

1991

complex [73, 258, 4485]. Surprises included the paucity of transcription units (only 6 genes in 100 kb of DNA) and the absence of introns [2891]. Ruiz-G´omez and Modolell used 74 terminal deﬁciencies with breakpoints in the AS-C for a deletion analysis [3688] and tested two hypotheses: (1) the “Differential Threshold Model,” where each MC needs a different amount of Ac or Sc, thus explaining why bristles are affected in a deﬁnite sequence as enhancers are whittled away; and (2) the “Site-speciﬁc Enhancer Model,” where each enhancer controls sc expression at a certain site. They favored the latter model, which turned out to be correct. The groups of equipotent cells that Stern predicted in 1954 were documented histologically by in situ RNA hybridization and named “proneural clusters” (PNCs) [1454, 3637, 3957]. In situ detection of Ac and Sc protein distributions allowed PNCs to be resolved in greater detail [912, 3982]. James Skeath and Sean

1992

1995

Carroll validated Stern’s fanciful idea of cryptic singularities by identifying an “inactive cluster” anterior to the dorsocentrals and by showing that derepressing the AS-C induces MCs there (their Fig. 5) [3982]. Skeath et al. found that when the AS-C is broken in half by an inversion, co-expression of Ac and Sc in (embryonic) PNCs ceases, and Ac and Sc become expressed in complementary subsets of clusters [3986]. The heretical implication was that ac and sc respond independently to PNC-speciﬁc enhancers, rather than controlling certain subsets of clusters via differences in their gene products [2721]. Jos´e Luis G´omez-Skarmeta et al. described the ﬁrst mutation that eliminates sc function without disabling any AS-C enhancers (sc M6 ) [1538], thereby allowing the role of sc to be assessed unambiguously for the ﬁrst time [628]. They also produced the ﬁrst enhancer map compiled via reporter gene constructs (Fig. 3.5). By these means, they showed that the “classic” roles that had been assumed

42

IMAGINAL DISCS

Macrochaetes

a % Present

100

0

mc

PSA PDC ADC 70

y

60

PNP 50

40

null point mutation

ac

ASA PS 30

sc

20

ANP APA 10

l’sc

PPA 0

PSC ASC -10

-20kb

T2

% Present

100

0

mc

PSA PDC ADC

b

70

60

PNP 50

40

ASA PS 30

20

ANP APA 10

PPA 0

PSC ASC -10

-20kb

Delete Detach y

ac

sc

l’sc

T2

% Present

100

0

c

mc

PSA PDC ADC 70 70

60

PNP 50

40

ASA PS 30

20

ANP APA 10

PPA 0

Delete Detach ac

sc

l’sc

T2

PSC ASC -10

-20kb

CHAPTER THREE. BRISTLE PATTERNS

for ac and sc were wrong. The old AS-C paradigm toppled, and a new one emerged, as recounted below. In the ensuing “gold rush,” many researchers have been picking apart each enhancer to ascertain what transacting factors inﬂuence it (e.g., see [1380]).

In 1989, Achaete and Scute were found to mark ‘‘proneural clusters’’ As discussed above, Stern deduced that each SOP arises from a group of equipotent cells, whose competence to respond to bristle-inducing signals is conferred by ac or sc. Because prepattern signals should be congruent with the ﬁnal pattern (except for cryptic singularities), the expression of ac and sc need not be conﬁned to bristles sites. In theory, ac and sc could be transcribed ubiquitously. In fact, they are not. In 1989, Romani et al. [3637] found that ac and sc RNA is restricted to nests of cells at future bristle sites. They called these nests “proneural clusters” (PNCs) [3950]. PNCs fulﬁll Stern’s 1954 prophecy. They were resolved more clearly when Ac and Sc protein distributions were seen in 1991 [912, 3982]. Each PNC has a characteristic size, shape, time of appearance, and time of disappearance [589, 912, 1538, 3637, 3982]. 1. Ac and Sc are detectable in each cluster before the SOP(s) appears. This order is consistent with the idea that Ac and Sc enable a cell to adopt the SOP state. The number of Ac- and Sc-expressing cells increases

43

2.

3.

4.

5.

6. 7.

until SOP emergence, as does the intensity of expression. Most clusters yield only one SOP. In those cases, the nest is roughly circular. Cells near the center typically manifest more Ac and Sc than those near the periphery, and one of the intensely expressing central cells becomes the SOP. Stern had imagined such a central “peak” based on his embryonic ﬁeld analogy [3613, 4096--4098]. At their zenith, most single-SOP nests contain ∼20– 30 cells. This size is roughly what was expected based on PDC shifts in ac mosaics. The PSA nest is exceptionally small (∼10 cells). The DC and SC clusters produce two SOPs each. In each case, the posterior SOP develops ﬁrst. The PDC arises at a clearly eccentric location within its nest [3373], as does the APA. Eccentric SOPs rule out certain models for how the SOP is selected. In each PNC, Ac and Sc reach higher levels in a subcluster of ∼6 cells. Among those few cells one cell attains a still greater level, which evidently boosts it above some sort of “ignition threshold” because it inevitably becomes the SOP [637, 918]. In nests that produce two SOPs, the subclusters that precede the SOPs are spatially and temporally distinct. The location of the SOP(s) within a PNC is virtually the same from ﬂy to ﬂy. Ac and Sc fade below the limits of detection before the SOP begins dividing. Presumably, the levels of Ac and Sc can wane because they have triggered the

FIGURE 3.5. Roles of achaete and scute in bristle development on the notum.

a. Phenotype of sc M6 , a sc null with a nonsense-codon mutation (“X”) before the HLH domain [1538]. Unlike previous nulls, this allele is not associated with deletions or inversions that confound gene-phenotype analyses (e.g., b, c). Bars indicate frequencies of MC presence. Sites are listed in enhancer order (dashed lines; cf. Fig. 3.4 for details and Fig. 2.6 for MC key; mc = microchaetes). Surprisingly, sc is not needed for bristles that were thought to depend on it (b), including scutellars (PSC, ASC) whence scute got its name [470]. b, c. “Classical” (but incorrect) roles of ac and sc as deduced from nulls that involve major DNA alterations. In each case, deletion of a gene (sc or ac) was achieved by crossing over between inverted chromosomes with breakpoints at different AS-C sites [2561]. With such complex “knock outs,” it is impossible to distinguish the effects of gene inactivation from the effects of disabling enhancers. Thus, the complementary sets of bristles affected by these constructs (b vs. c) give a false impression of the relative roles of sc and ac (cf. a). b. Phenotype of a sc null , In(1)sc8L sc4 R , where L and R denote left or right halves of inversions contributing to the recombinant. The deletion removes sc and the PNP enhancer, and the inversion moves proximal enhancers ASA, etc., away from achaete to the other (heterochromatic) end of the chromosome. Thus, the only enhancers still able to inﬂuence ac are mc, PSA, PDC, and ADC. Other PSA and ADC enhancers must exist in the deleted or detached domains (probably in the sc promoter region [2721]) because these bristles are often missing. c. Phenotype of an ac null , In(1)y3 P L sc8 R , which removes ac and enhancers for PSA, PDC, and ADC. Microchaetes may be only partly affected because their enhancers extend past the deletion [3688]. Although proximal enhancers PNP, etc., are disconnected from ac by the inversion, they remain linked to sc and therefore can apparently still function normally. All data are from [1538]. N.B.: The yellow (y) gene has its own set of cis-enhancers [1442, 1443], and mutations therein produce a menagerie of pigment patterns [1435, 3055] (as variegated as dog or cat breeds), suggesting that some of the same spatiotemporal trans-factors that control the AS-C may also regulate yellow [3056].

44

IMAGINAL DISCS

next step in SOP differentiation. The SOP must be able to regulate its levels of Ac and Sc separately from other cells of the nest because the SOP typically stops expressing Ac and Sc at a different time (earlier or later) from the remaining cells. PNC cells are arrested in the G2 phase of the cell cycle [4427] and are recognizable by higher levels of Cyclin A [2225]. Mitotic quiescence appears essential for proper selection of SOPs because premature entry into mitosis (prompted by artiﬁcial activation of string) disturbs SOP emergence [2225]. As for why cells must stop dividing to select a SOP, it is possible that apical signaling is hindered when dividing cells retract basally [3823], and the same logic may apply in the morphogenetic furrow where synchrony is also imposed [1126, 1903]. Quiescence cannot be under AS-C control because ac− sc− discs have G2-arrested clusters like the wild-type [4427]. Arrest is probably enforced by a parallel circuit: the same trans-acting factors that affect the AS-C may bind directly to a separate set of cis-enhancers at the string locus [2480], where they can suppress this mitosislicensing agent without needing Achaete or Scute. The discovery of these pools of nonmitotic cells answered the old question of why bristle vs. non-bristle lineages appear to diverge so early in development [996, 1372, 1742]: the split is not due to compartment-like lineage restrictions, but rather to withdrawal of pre-bristle cells from the cell cycle long before cell types are ﬁnalized. The above observations provide clues about how SOPs are selected. Before trying to ﬁt these puzzle pieces together, however, additional clues at the genetic level are examined below.

In 1995, the old AS-C paradigm toppled and a new one emerged The old AS-C paradigm was based mainly on breakpoint-associated ac LOF and sc LOF alleles [1360, 1453]. Given the complementarity of their phenotypes (Fig. 3.5), the expectation was that “ac-dependent” bristle sites would mainly use Ac, and “sc-dependent” sites would mainly use Sc. It thus came as a shock when ac and sc were found to be expressed together in PNCs [637, 3530, 3637]. To explain this correlation, a mutual activation (of ac by sc and of sc by ac) was invoked and a two-step process was imagined [2721, 3982, 4453]. In Step 1, separate enhancers would turn ac and sc on in complementary sets of clusters, with each site using one or the other gene as its founder. In Step 2, Ac would trans-activate sc and Sc would trans-activate ac in each cluster. Although parsi-

monious, this idea turned out to be wrong with regard to PNCs (although it does apply to SOPs, as explained below). In 1995, G´omez-Skarmeta et al. disproved this “Mutual Activation Model” and solved the Bristle Coding Enigma of the AS-C [1538]. To knock out sc function, they used a nonsense mutation (sc M6 ) that differs from all prior sc and ac nulls insofar as it does not disrupt any surrounding AS-C enhancers. Thus, it revealed the actual bristles controlled by sc – a subset that happens to differ dramatically from the sc-dependent subset as classically deﬁned (Fig. 3.5). The Mutual Activation Model predicts that Ac should only be found at acdependent sites in a sc null . In fact, Ac accumulates in all PNCs of sc M6 ﬂies. Additional disproof of the model came from inversions that abolished ac-sc coexpression by merely severing the connections between certain enhancers and either ac or sc [1538, 3984, 3986]. Therefore, coexpression is due to shared enhancers that activate both genes per cluster (cf. point 3 below), not to stimulation of ac by sc or vice versa. Such a possibility had been suspected [2062] but not tested rigorously. E(spl)C genes rely on the shared-enhancer strategy to a much more limited extent [871, 3075]. This “AS-CEpiphany” of 1995 has forced us to rethink how anatomy is controlled by ac and sc. The old and new conceptual frameworks are contrasted below: 1. The reason breakpoint-associated ac LOF and sc LOF alleles (vs. sc M6 ) remove certain subsets of bristles is not because they affect one or the other gene per se. (Indeed, ac and sc are functionally interchangeable [191, 918, 948, 3628], so different bristles cannot rely on qualitatively different Ac vs. Sc proteins.) Rather, allelic speciﬁcity is due to deletion or detachment of speciﬁc AS-C enhancers, each of which affects a definite body region (Fig. 3.5). For example, ac1 removes the PDC because the PDC enhancer is deleted (Fig. 3.1), not because the ac gene is defective, and the sc M6 phenotype shows that this enhancer does not need sc. 2. Categories of bristles [1802, 2561] and sensilla [948, 2519, 3637] that were formerly seen as being “ac dependent” or “sc dependent” must now be viewed as being governed by enhancers that happen to lie near ac or sc, respectively. For example, the scutellars were thought to be controlled by sc, but the sc M6 phenotype indicates that they are more dependent on ac (Fig. 3.5). 3. Temporal differences that were ascribed to sc vs. ac

CHAPTER THREE. BRISTLE PATTERNS

(e.g., sc-dependent MCs preceding ac-dependent mCs [1741], and sc-dependent chemosensory bristles preceding ac-dependent mechanosensory bristles [1802]) are probably due to “heterochronic” enhancers that are activated at different times [4584, 4844]. 4. The reason no model ever succeeded in parsimoniously explaining the allele-speciﬁc bristle subsets is that the order of enhancers along the AS-C is scrambled relative to the MC pattern [1453, 1538] (Fig. 3.4). There is no “MC homunculus” analogous to the “homeobox homunculus” [1805] that obeys the order of body segments [1113, 2508, 2672, 3494]. In this regard, the AS-C resembles the hairy locus, where enhancers for pair-rule stripes 1–7 (anterior to posterior) are scrambled: (3 & 4), 7, (6 & 2), 5, 1 [2376] (cf. similar chaos at even skipped [1323, 3717, 3992] and the lack of a “muscle homunculus” in mice [2336]). 5. The phenomenon of competence does not involve reception of an external inductive signal at the cell surface (Fig. 3.2). Rather, it entails AS-C enhancers that directly respond to intracellular position-speciﬁc, trans-acting factors. Because the enhancers (not the genes) are responsible for the competence, ac and sc are more properly called “proneural” than “competence” genes. Operationally, this distinction is supported by the ability of generalized ac or sc overexpression to cause extra MCs at abnormal sites [191, 635, 1348, 3628]. Such a result would not be expected if the genes merely enabled cells to respond to pre-existing signals at ﬁxed locations. 6. Now that the old puzzle of why speciﬁc ac LOF or sc LOF alleles remove certain bristles has been solved, the remaining question is how AS-C enhancers are controlled by spatial or temporal cues that are upstream in the genetic hierarchy [4584]. Much progress has been made in identifying these “prepattern” factors, and these ﬁndings are discussed in Chapters 5 and 6. Not all enhancers activate ac and sc equally, because only certain MCs are missing in the sc null point mutant (Fig. 3.5). This favoritism may be due to the kind of cognate lock-and-key afﬁnities between enhancers and promoters that are found at the adjacent yellow locus [1534], in the Bithorax Complex [275, 1821, 3975], and elsewhere [349, 2194, 2537, 2827, 3180, 3833]. In other words, transcription factors that bind certain enhancers (for sites that lack bristles in sc M6 ) would have greater afﬁnity for the sc promoter than for the ac promoter. Indeed, the sc and ac promoters differ appreciably from one another [918, 3171]. Such speciﬁcity would also explain why l’sc is

45

silent in SOPs, despite its closer proximity than ac or sc to some MC enhancers (Fig. 3.4) [3504]. Alternatively, these preferences could be mediated by factors that bend AS-C DNA into knotlike conﬁgurations where contacts are sterically possible only at certain points [275]. One candidate for such a role is the product of Dichaete. Dominant mutations in this gene have long been known to cause scLOF -like patterns of missing bristles [3405, 4178]. Dichaete is a Sox-domain protein that kinks DNA into an 85◦ angle [2635, 3707], so it might constrain the contacts between trans-acting factors (bound at cis-enhancers) and the basal transcription apparatus (bound at core promoters) [317, 1480, 1629, 3360], depending on where its binding sites are located within the AS-C. However, Dichaete cannot be a general partner in AS-C function because it is not normally expressed in wing discs [2977]. A more likely candidate there is Chip [3504], which ties DNA into loops [3600, 4456]. Still another suspect is the gargantuan Mediator complex [134, 412, 4386], which is known to bridge enhancers to promoters in other systems [1703, 2488, 2665]. The sc M6 phenotype suggests that some bristles need more Sc than Ac protein, but this cannot be true because sc and ac are functionally redundant [952, 2019, 2891]. Rather, the sum total of both proteins must dictate whether a bristle forms. If that total exceeds the threshold, then a bristle will arise. Otherwise, it will not.

Proneural ‘‘spots’’ shrink to SOP ‘‘dots’’ How is a SOP chosen within a PNC? The deciding factor seems to be the amount of Ac and Sc. As stated above, the intensity of AS-C proteins is uneven within each cluster, and the cell with the most Ac and Sc inevitably becomes the SOP [912, 1538]. The dose dependence of the selection mechanism is conﬁrmed by mosaics where ac+ sc+ (full-dose) cells become SOPs 2–4 times more often in a background of ac− sc− /++ (half-dose) cells than in a background of ac+ sc+ cells [912, 1794]. Why does one cell acquire more Ac and Sc than other cells in its cluster, and why does the SOP tend to arise at a certain site in each PNC? Genes that are hierarchically upstream of the AS-C (e.g., u-shaped and pannier [911, 1671]) are known to impose spatial biases [3966], but those biases can ﬁt several sorts of scenarios: 1. “Predestined SOP Model.” SOP sites could be ﬁxed by antecedent patterns of expression by genes that are hierarchically upstream of the AS-C (cf. Ch. 6). Sharp expression boundaries could pinpoint SOP sites within PNCs by their intersections, but whether

46

IMAGINAL DISCS

SOP

Proneural Cluster

Inhibition

Lateral Inhibition Model Mutual Inhibition Model

proneural cell Delta

Inhibition

proneural cell

Delta

Notch

AS-C

AS-C Delta

Delta

a

Notch

b c

there are enough boundaries to designate all sites is not known. Also unknown is whether such precision is attainable by diffusible signals [210, 902, 903, 2515, 3584], given the formidable task of addressing a one-cell target in a “forest” of tall, thin epithelial cells. 2. “Contest Model.” PNC cells might compete to become the SOP, perhaps via Dl-N “shouting matches” (Fig. 3.6; see below) [178]. Because SOPs arise within predictable subclusters, the competition must be biased [3954]. The bias could be spatial (e.g., proximity

Notch

AS-C

AS-C Notch

epidermal cell

SOP

d

to a diffusible activator or inhibitor of the AS-C) or temporal (e.g., starting to make Ac and Sc earlier). Accuracy need not be as great as in the previous model because a single victor will emerge as long as there are ≤6 contiguous contenders (i.e., the biasing process need only delimit 6-cell spots). 3. “Diffusible Activator Model.” If all PNC cells were to secrete a diffusible activator of the AS-C, then its concentration should intensify in the middle of the PNC and a cell there would be ﬁrst to cross the

CHAPTER THREE. BRISTLE PATTERNS

47

FIGURE 3.6. Hypothetical mechanisms for ensuring a single bristle per proneural cluster.

In the Lateral Inhibition Model (above), the SOP prevents neighbors from becoming SOPs [4095]. Arcs denote inhibitory signals that could be transmitted by diffusion or contact. Contact between nonadjacent cells would require extensions like the ﬁlopodia seen on moth scale cells [3051]. In the Mutual Inhibition Model (below), every proneural cell inhibits adjacent cells, and the SOP can be chosen later [1563, 3022, 3270]. Uncommitted proneural cells are shaded, the SOP is black, and cells that succumb to inhibition (and become epidermal) are white. a, b. Supposed interactions at different stages, according to the Mutual Inhibition Model. Cubes are individual cells, and inscribed circles are nuclei. a. Initially, all cells are equivalent in the extent to which they emit (Delta ligand) and receive (Notch receptor) inhibitory signals, but this balance is unstable because each cell’s AS-C feeds back on itself in a loop that traverses the other cell [1458]. The feedback is positive because the loop has two negative steps. (Arrows denote activation; cross-barred “ ” lines denote repression.) Any asymmetry will be ampliﬁed, so if one cell is slightly more inhibited, its AS-C and Delta activities will wane, as will its ability to inhibit. Ampliﬁcation is damped by a separate loop that uses the EGFR pathway (not shown) [917]. Other factors may also prevent cells from stably expressing both Notch and Delta [990, 2839]. b. Eventually this cell shuts off its AS-C and becomes epidermal. Conversely, the cell that is less inhibited keeps increasing its AS-C activity and, at some point, crosses a threshold that triggers an autocatalytic loop, which keeps the AS-C switched on. By this stage the “winner” has purged its Notch receptors, and the “loser” has purged its Delta ligands. c, d. Two of the possible outcomes for a 5-by-5 array of cells undergoing mutual inhibition [1614, 1805, 4269]. (Another is parallel stripes.) The intended outcome, a single SOP among 25 cells, is actually impossible. One way out of this dilemma is to imagine that the inhibitory arc of the loop is functional, but the excitatory arc is disabled. In that case, cells would “damp” one another but would not become SOPs, based on how severely they themselves are damped. Another means of biasing the SOP decision would then be needed, perhaps involving underlying prepattern factors or an overlay of Extramacrochaetae protein [1458]. This montage is adapted from [1458, 3866, 4115] (a, b) and [1181, 1614, 1889, 4269] (c, d). N.B.: In the wing, Delta-Notch signaling manages to create 3-cell wide stripes that become veins [989] by a feedback mechanism that seems similar [1951] but may not be (cf. Ch. 6). Thus, the same circuit may be used in different ways in different contexts [444, 627, 1457, 2018]. Odd results in the eye have raised questions, however, about the circuit’s versatility and validity [184]. See also App. 7.

“ignition threshold” to become the SOP [1462, 3584]. This scheme ﬁnds support in the intermediate level of Ac-Sc (a pre-SOP state? [912]) in subclusters where SOPs arise, but eccentric SOPs are problematic. The ﬁrst two schemes are actually components of larger hypotheses (the Lateral vs. Mutual Inhibition Models [4480]) that are discussed later. They are being treated here as stand-alone models to contrast their premises and predictions. The Predestined SOP Model is hard to reconcile with the ability of ac1 clones to displace the PDC within its cluster, because only the ordained cell should be endowed with enough Ac or Sc. Perhaps it is the highest relative (vs. absolute) level of Ac or Sc that determines the SOP. Cells within a cluster might be comparing their levels of Ac or Sc via Dl-N signaling. However, the decision cannot be purely relative because raising the absolute level of Ac or Sc can evoke extra SOPs per cluster [191, 635, 3628]. The Contest Model implies that close matches should sometimes arise in which two rivals both attain an advanced state of SOP commitment before one retreats to the default epidermal state. Indeed, pairs of SOPlike cells are occasionally seen in PNCs that ultimately

make only one bristle [1925]. How such disputes might be resolved to yield a single SOP is discussed later. The Diffusible Activator Model could explain eccentric SOPs if those nests have more of an AS-C inhibitor (e.g., Extramacrochaetae) centrally than peripherally, thus forcing the SOP to arise near the edge (like PDC displacements seen in ac1 mosaics). Some kind of diffusible factor is likely because mosaics for AS-CLOF [807, 3613, 4095, 4181] and AS-CGOF [1577, 4097] mutations often violate the rule of autonomy when the mutant/wild-type boundary skirts a bristle site. In those cases, mutant cells acquire the ability to make bristles, probably by diffusion of a rescuing agent from nearby wild-type cells [1462, 4098]. It is worth stressing that it is close neighborhood [which causes the nonautonomy] unrelated to total extent of the ac1 area which may appear as a small island just covering the bristle site or a large patch whose border happens to pass by it. This suggests a simple spread of ac+ dependent material into the ac1 tissue patch. This material then nonautonomously endows the ac1 cells with the ability to respond to the prepattern by differentiation of a bristle if only of rudimentary size. It seems to be a case of substitution therapy equivalent to the presumed support in mosaics of genedeficient cells by nondeficient tissue. [4098]

48

The rescuing molecule cannot be Ac or Sc, because they localize to the nucleus [912]. The soluble agent, which appears to be cleaved from a transmembrane precursor [3640], is probably Spitz [917] – a ligand for the EGFR pathway (cf. Ch. 6). That pathway participates in a positive feedback loop with the AS-C (see below): AS-C EGFR AS-C [917].

The SOP uses a feedback loop to raise its Ac and Sc levels Although ac-sc cross-activation was disproven for PNCs, it apparently does occur in SOPs [918, 1538, 2721]. When a 3.7-kb piece of DNA immediately upstream of the sc transcription unit is joined to a lacZ reporter, this construct is found to be expressed in SOPs but not in any other cluster cells (except at DC and PSA sites, which must have enhancers in this fragment [2721]). The same is true for a construct of the 0.8-kb ac promoter joined to lacZ. Both of these transgenes are silenced in an ac− sc− background unless ligated to PNC enhancers, implying that ac and sc genes of SOPs require Ac or Sc for autoor cross-activation. These feedback loops apparently must be primed to a threshold before they sustain themselves [917], and PNC enhancers supply the needed boost [918]. Earlier studies showed expression in all PNC cells (not just SOPs) of lacZ reporter genes driven only by the ac or sc promoter (sans PNC enhancers) [2721, 4453], but those results may have been complicated by staining artifacts or deletion of control sites that restrict expression in vivo [918, 1538]. Ac and Sc belong to the class of bHLH transcription factors that bind E boxes (Fig. 2.4). There are three E boxes in the sc promoter [918] and four in the ac promoter, although one is unresponsive to proneural proteins [3171, 4452]. The “SOP enhancer” for sc has been whittled down to a 356 b.p. piece (∼2.7 kb from the transcription start site) [918], and only one E box appears essential for auto-activation. These results imply the positive feedback loop “more {Ac and Sc} bind E boxes more {Ac and Sc},” but if this loop automatically raises Ac-Sc levels high enough to spark SOP differentiation [1538], then what prevents non-SOP cells in each PNC from doing the same? Conceivably, access of proneural proteins to E boxes in nonSOP cells is blocked by trans-acting repressors that are expressed in all PNC cells except the SOP [918, 1538]. One such candidate – Extramacrochaetae – is discussed later. Another candidate is encoded by the klumpfuss (klu) gene [2250]:

IMAGINAL DISCS

1. klu is switched on in PNCs when Ac becomes detectable but is switched off in the SOP as soon as it arises. Thus, klu’s expression could allow it to distinguish non-SOP cells from SOPs. 2. klu is genetically downstream of the AS-C (kluLOF is epistatic to acGOF in causing bristle loss), but its transcription in PNCs is independent (klu still turns on in ac− sc− ﬂies). The agents that activate klu are unknown. 3. Klu has four zinc ﬁngers and other hallmarks of a transcription factor, so it could implement SOP vs. non-SOP states directly at the DNA level. 4. Despite the above features, klu cannot be a simple selector gene because its effects appear to be nonautonomous: SOPs do arise in kluLOF mutants (as judged by high Ac levels) but fail to complete bristle development. Thus, klu causes bristle loss via an effect on a cell (SOP) that does not express klu. However, there is an autonomy-based alternative: the cell that becomes the SOP might need klu to ﬁrst turn on (leaving a legacy of other gene settings) and then off to launch the SOP on its (kluindependent) differentiative path. The AS-C must act differently in SOPs vs. non-SOPs because ubiquitous overexpression of an antagonist (hairy) quashes Ac in all proneural cells except SOPs [3982]. Further evidence for distinct (SOP vs. non-SOP) regulation is given below regarding the E(spl)-C.

Two other bHLH genes (asense and daughterless) assist SOPs A third bHLH gene in the AS-C – asense (ase) – also participates in bristle development. Like ac and sc, it is transcribed in SOPs, but unlike them it is not transcribed in other PNC cells (except on the wing edge) [438, 1079, 2039]. Its SOP expression appears late in cluster development, suggesting that it is turned on by high levels of Ac and Sc. Indeed, there are four E boxes in the 5 untranslated region of the ase transcript [918, 2039], and ase expression disappears in an ac− sc− background in all SOPs (except for the wing edge) [438, 1079]. However, ase is dispensable for most bristles, perhaps because other (AS-C?) genes compensate for its absence. In asenull ﬂies, only the wing [2039] and tergites [2690] seem affected. The stout bristles on the wing margin are often stunted, fused, or twinned, but rarely absent – implying a function for ase in bristle differentiation [1079, 2039], although the partial loss of tergital mCs suggests a role in SOP inception as well.

CHAPTER THREE. BRISTLE PATTERNS

HLH proteins typically prefer to homo- or heterodimerize with only certain partners, which in turn dictates preferences for particular DNA-binding sites [68, 348, 2089, 3017, 3179]. For example, Ac and Sc were each found to require a binding partner from outside the AS-C in order to function [588, 589, 4452, 4453]. That partner is Daughterless. As its name implies, the daughterless (da) gene was originally identiﬁed by its LOF effects on sex determination. Only later was its involvement in bristle development discovered. Flies that are doubly heterozygous for a danull allele and an AS-C deﬁciency lack some MCs, while heterozygotes for either lesion alone look wild-type [949]. This dose-dependent interaction is attributable to a need for Ac/Da and Sc/Da heterodimers, which have been shown to form in vitro [588, 589, 918, 4452]. Unlike Ac and Sc, Da is expressed ubiquitously in discs at a level that is no higher inside bristle-yielding PNCs than outside them [905, 4435]. Based on its role in the embryonic PNS, Da seems to be required for the SOP to divide [2019]. Speciﬁcally, da appears to activate cell-cycle genes in the SOP after it emerges from the cluster [949, 1755]. One downstream gene regulated by da may be Cyclin A (CycA): a CycALOF allele that causes missing MCs was induced by a P-element insertion in CycA’s ﬁrst intron, and that intron contains ﬁve E boxes [4415].

‘‘Lateral’’ or ‘‘mutual’’ inhibition ensures one SOP per PNC As mentioned earlier, Stern drew two inferences from PDC displacements in ac1 mosaics: (1) each MC SOP comes from a group of equipotent cells, and (2) the SOP inhibits its neighbors from adopting its fate [4095, 4098]. The ﬁrst conjecture was validated by the discovery of PNCs. The second was later formalized as the “Lateral Inhibition Model” (Fig. 3.6) [112, 1458, 1808, 3207, 3950]. This model made several predictions that were also supported by subsequent observations: Prediction 1:

Prediction 2:

Killing an incipient SOP should remove the inhibition and allow a different cell to become the SOP. Disabling the inhibitory mechanism should allow all the cells in a PNC to become bristles.

The ﬁrst prediction was tested in the embryonic CNS of grasshoppers, whose neuroblasts arise somewhat like SOPs of the ﬂy PNS [587, 1071, 1073, 1745, 1804]. Ablating an incipient neuroblast does indeed cause a nearby

49

cell to change its fate (from ectodermal to neural) to replace the dead one [1072, 4231]. This ability of PNCs to regenerate a progenitor cell provides direct evidence for the Lateral Inhibition Model. Consistent with the second prediction, LOF alleles of various genes cause tufts of bristles at existing MC sites (and high densities in mC zones). Most of these genes are in (or associated with) the Notch pathway [114], including Notch [1742, 1797, 3891], Delta [3277, 4859], Su(H) [3820, 3826], E(spl)-C bHLH genes [991, 1794], groucho [1794], kuzbanian [3640, 4025], shibire [1802, 1803, 3425], and big brain [1075, 3519]. Among the foregoing, big brainLOF has milder effects (even as a null), as do GOF alleles of Hairless [200, 2657] – a Su(H) antagonist. GOF alleles [191, 635, 1348, 1802] and artiﬁcial overexpression [1854, 3027, 3628] of proneural genes also cause extra bristles, but these phenotypes differ in several ways from the NotchLOF tuft syndrome [2959, 3950]: 1. Ectopic bristles arise in areas that are normally bare. 2. MCs remain separated by intervening epidermal cells. 3. Increases in mC density are less severe. The ability to put bristles into new areas (e.g., the ﬂanks of the abdomen) underscores the role of proneural genes in conferring neural competence to speciﬁc body regions [1804]. Notch-pathway genes do not share this role, with one exception. LOF alleles of groucho cause extra bristles in the wing blade and scutellum – the same areas affected by LOF alleles of hairy [1794]. The explanation is that Groucho is a promiscuous corepressor that interacts with Hairy, but not via the Notch pathway [1794, 3278]. The milder MC and mC phenotypes seen in AS-CGOF mutants [191, 1577, 1802] imply that lateral inhibition still functions, whereas it is disabled in Notch-pathway LOF mutants. Herein lies the key difference between AS-C and Notch-pathway genes. The idea that bristle cells prevent nearby cells from becoming bristles was ﬁrst proposed in 1940 by Vincent Wigglesworth [1145, 4657]. The hemipteran insect that he studied – Rhodnius prolixus – bears evenly spaced bristles on its abdomen [4663]. By comparing bristle positions in successive instars of a single individual, he found that the new bristles are added in the largest gaps of the old pattern [4657]. To explain this trend, he proposed that bristle cells deplete their vicinity of a diffusible factor that is needed for bristle initiation. This depletion prevents any new bristles from arising within a certain range of any extant bristle. Only when growth

50

pushes bristles far enough apart should an intervening cell become a bristle in the next instar. This “Local Depletion Model” is formally equivalent to the Lateral Inhibition Model, where bristle cells emit an inhibitor instead of consuming an inducer [3207, 4660]. These exclusionary (“inhibitory”) ﬁelds are thought to serve two different roles in Drosophila: 1. Fine-tuning: Inhibitory ﬁelds ensure one bristle per potential site. 2. Bristle spacing: Inhibitory ﬁelds place bristles a minimum distance apart. In more recent years, an alternative hypothesis – the “Mutual Inhibition Model” – has gained popularity [1563, 1797, 3022]. It asserts that every proneural cell (not just the SOP) inhibits its neighbors via Dl ligands. Inhibited cells reduce AS-C activity so low that they cannot become SOPs (Fig. 3.6), except for one cell (the future SOP), which somehow escapes inhibition. Like the C. elegans scheme that inspired it [3866], this model is usually depicted as a contest between equivalent cells, which adopt alternative fates after one wins and the other loses [1794, 4115]. If groups of ∼25 cells compete, then multiple SOPs should arise in checkerboard, stripe, or ring patterns (Fig. 3.6) [1889, 4269]. How can a single-SOP outcome be mandated? One way would be for the contests to occur in only a core subcluster (∼6 out of the ∼25 cells). Alternatively, the SOP could be shielded from inhibition by boosting its AS-C above a threshold for autocatalysis [443, 1458, 3022, 3823]. The two models (“LI” vs. “MI”) are debated below. Like the Contest Model discussed above, these schemes invoke cellular chitchat, but they are more concerned with preventing excess SOPs than with SOP selection per se.

Notch-pathway and proneural genes are functionally coupled The MI model envisions a feedback loop between Notch-pathway and proneural genes (Fig. 3.6). This same loop could mediate inhibition in the LI model. Some kind of cell-autonomous “N Dl” connection must exist because cells with excessive N-intra signaling in their cytoplasm lose Dl from their surface [3270]. Moreover, proneural-Notch cross-talk must exist because haplo-insufﬁciency for da enhances NLOF (split) eye phenotypes [437]. Genes wired to the end of the Notch pathway (Dl N Su(H) E(spl)-C targets?) will be repressed because E(spl)-C bHLH proteins recruit the Gro corepressor, whereas genes affected by Ac/Sc/Da should

IMAGINAL DISCS

be activated (Ac/Sc/Da targets?). If Dl were an AS-C target and the AS-C were an E(spl)-C target, then the loop would be complete. Both links appear to be functional, as the following evidence argues. However, it is important to keep in mind that showing a link under one set of circumstances does not prove that the link is used at a physiologically meaningful level in other cell types at other times [113, 871, 1330, 4584]. 1. Evidence for an “AS-C Dl” link: a. GOF: In wing discs, Dl is expressed in PNCs [2296], and ectopic expression of sc (or l’sc) activates Dl transcription in ectopic congruent areas [1854]. The transcription factor Senseless (zinc-ﬁnger class) has been implicated as a mediator of this link [3127]. b. LOF: Deleting the AS-C abolishes Dl expression in PNCs of the embryonic CNS [1672]. c. LOF: Mutating the binding sites for Ac/Da heterodimers in the Dl promoter reduces expression of a lacZ reporter gene (driven by that promoter) in PNCs of the embryonic neuroectoderm [2359]. 2. Evidence for an “E(spl)-C AS-C” link: a. GOF: In cultured cells, expression of m8 and m5 reduces the ability of Da to activate transcription of a CAT reporter gene (driven by an m8 promoter fragment that has a Da-binding site) [3171]. b. GOF: Overexpression of m8 (via a UAS-m8 transgene driven by da- or hs-Gal4) prevents SOPs from arising [3037, 4256] – an effect traceable to reduced sc transcription in SOPs (mild m8GOF ) [918] or PNCs (extreme m8GOF ) [991]. Also, bristles fail to develop when m8 is overexpressed in SOPs (UAS-m8 driven by scabrous-Gal4) [3037]. c. Correlation: E(spl)-C bHLH proteins are naturally absent from the SOP (high AS-C level) but present in the rest of the PNC (low AS-C level) [2052]. In theory, the “E(spl)-C AS-C” effect could be mediated in at least three ways [1458, 2063, 4318]: 1. Protein-protein binding via HLH domains. E(spl)-C bHLH proteins could trap Ac, Sc, or Da monomers in inert heterodimers [68, 348, 1484]. 2. Transcriptional repression of AS-C genes by DNA binding. 3. Transcriptional repression of genes that are downstream targets of AS-C proteins. The ﬁrst idea was discounted because M8 does not block Ac/Da or Sc/Da dimer formation in vitro [4452], although M8 can bind Ac, Sc, and Da under other conditions [68, 1484]. The second route seems likely for “m8 ac.” Although M8 does not bind E boxes [4452], it does bind N

CHAPTER THREE. BRISTLE PATTERNS

boxes (Fig. 2.4) [3171, 4318], and there is an N box in ac’s promoter. Indeed, m8 reduces transcription of an acpromoter CAT reporter [1794]. The “m8 sc” link should work the same way because there is an N box in sc’s “SOP enhancer” [918], but M8 still represses sc when this box is deleted. The reason seems to be that M8 can also get to the enhancer indirectly by binding an NF-κB-like factor that, in turn, binds a motif near the N box [918]. Convincing evidence for a transcriptional mode of action for M7 comes from a clever “chimera” experiment. E(spl)-C bHLH proteins are repressors mainly because the WRPW motif at their C-terminus recruits the co-repressor Groucho (Fig. 2.4) [1794]. When an artiﬁcial “m7ACT ” gene was built by replacing WRPW with a VP16 activator domain (from herpes virus [4394, 4433, 4483]) [2063] and put into ﬂies with a Gal4 driver, it caused extra bristles in various body regions – an effect opposite to the balding seen when native E(spl)-C bHLH proteins are overexpressed [918, 991, 3037, 4256]. It also caused ectopic activation of lacZ reporter genes linked to 4-kb promoter fragments from ac and sc. Apparently, m7ACT – and, by inference, native m7 – acts by inﬂuencing the AS-C at the transcriptional level (Possibility 2). The effect is apparently not exerted via E boxes because M8 does not displace AS-C/Da heterodimers from E boxes in vitro [4452]. The existence of E boxes in m8’s promoter (cf. Fig. 2.4) [171] suggests an “AS-C E(spl)-C” link [2317, 3171], and E boxes in m7 ’s promoter are required for reporter gene expression in PNCs [3974]. However, this connection is probably moot because 1. E(spl)-C bHLH genes are downregulated by N LOF , despite a functional AS-C [2052]. 2. E(spl)-C bHLH genes are activated by N GOF , even in an ac− sc− background [2052]. E(spl)-C” link were to exist, then Indeed, if an “AS-C the SOP’s abundant AS-C proteins should cause its level of E(spl)-C proteins to be the highest in the PNC, but the opposite is true: the SOP is the only PNC cell that lacks E(spl)-C bHLH proteins [2052]. The loss of these proteins from SOPs is attributable to a separate (indirect) path that is triggered by high levels of AS-C gene products: “AS-C senseless E(spl)-C” [3127]. According to the MI model, the loop between the AS-C and Notch should allow the AS-C loci of adjacent cells to interact [436, 1458, 1794]. In tracing the circuit from one cell’s AS-C into the other cell and back again (Fig. 3.6), there are two negative steps (E(spl)-C AS-C) so the overall loop is positive [1458]. For equivalent cells, the AS-C levels will be unstable. Under these conditions,

51

any stochastic change should “boomerang” (“higher AS-C loop even more AS-C” or “lower AS-C loop even less AS-C”), breaking the symmetry and tipping the balance. At some point, the victor’s AS-C level will cross a threshold, after which its autonomous SOP loop (Ac/Sc bind E boxes more Ac/Sc) will take over. Other facts are consistent with this scenario: 1. Heat-pulsing ﬂies carrying a t.s. N LOF allele upregulates sc in PNCs via an effect on sc’s “SOP enhancer” [918]. Similar stimulation of ac is seen in embryos that are defective in Notch-pathway genes [3983]. 2. In Su(H) null wing discs, ac is upregulated in PNCs [3820, 3827], supposedly via downregulation of the E(spl)-C. Interestingly, Dl is also upregulated – an effect consistent with the proposed feedback loop. Nevertheless, the circuitry inside SOPs must differ from that of ordinary PNC cells. 1. Deleting all 3 Su(H)-binding sites in the m8 promoter turns off m8 in PNCs (assayed by an m8 promoter-lacZ reporter) but turns it on in SOPs [2453]. Is the E(spl)-C regulated by genes outside the Notch pathway, and, if so, why should m8 be upregulated when deprived of activation by Su(H)? Conceivably, the deletions remove binding sites for unknown trans-acting repressors that are speciﬁc for SOPs (e.g., Senseless? [3127]). Deleting N boxes in m8’s promoter has the same effect in embryos: expression stops in PNCs and starts in neuroblasts [2317]. 2. PNCs arise normally in H null ﬂies, but SOPs fail to appear [197]. This balding is suppressed when E(spl)C bHLH genes are also deleted [197], which is strange because these genes are not normally expressed in SOPs [2052]. Possibly, H prevents Su(H) from turning E(spl)-C genes on in SOPs, but (for unknown reasons) has no effect in other PNC cells. Disabling H would cause “Su(H) E(spl)-C AS-C” (and stiﬂing of SOPs), but additionally disabling E(spl)-C would restore AS-C function [197]. These results have been interpreted as showing that the SOP is sensitive to inhibition from non-SOP cells [197] – a conclusion that would invalidate the LI model. However, the H null and double-mutant traits can also be explained by effects solely inside the SOP (given the circuitry above and the H Su(H) antagonism discussed in Ch. 2), so the facts do not inherently favor either model. (A similar restoration of normalcy is seen in the H LOF Su(H) LOF double mutant [3827].) Moreover, H transcripts are uniform in the epidermis when SOPs emerge [200], so it is unclear how H could be acting differently in SOP vs. non-SOP cells [3827].

52

3. The zinc-ﬁnger transcription factor Senseless (Sens) accumulates only in SOPs [3127]. Because (1) E boxes reside in the sens promoter and (2) binding sites for Sens (AAATCA) exist in the cis-regulatory regions of ac and sc (as well as m8), Sens could be mediating the positive feedback loop that fosters the surge of proneural proteins in SOPs: “AS-C Sens AS-C”. Indeed, sens exhibits strong synergy (i.e., dense tufts of bristles) with scute when they are co-expressed (via dpp-Gal4) in eye discs [3127]. 4. Overexpression of the EGFR inhibitor Argos suppresses SOPs without affecting PNCs [917]. It does so by blocking an autocrine loop: “AS-C EGFR ASC”. The blockage prevents the SOP from exceeding the level of Ac or Sc that is present in the PNC. The external ligand in this loop appears to be Spitz [917].

Doses of Notch-pathway genes can bias the SOP decision As stated above, SOP selection depends on AS-C dose in mosaics: a wild-type cell is 2–4 times more likely to become a SOP than a cell with half that dose (ac− sc− / ++) [912, 1794]. When the boundary between high- and low-dose cells bisects a PNC, the two genotypes presumably compete within the mosaic PNC, and their abilities to attain the SOP state are reﬂected in the overall frequencies of differently marked bristles. Such contests are a key part of the MI model, but they can also ﬁt the LI model. Maybe cells with more Ac and Sc are simply getting a head start in the race to become a SOP [917]. Such a temporal advantage might explain why female cells become SOPs 4 times more often than male cells at gender boundaries in gynandromorphs [1800]. Considering the ties between Notch-pathway genes and the AS-C, it is not surprising that doses of those genes also affect SOP choice. Notch, Delta, and Hairless are among the 21 ﬂy genes (excluding Minutes) that are haplo-insufﬁcient [118]. In haploid dose, both Notch and Delta cause more mCs [997, 4466], while a haploid dose of Hairless stiﬂes some MCs, reduces the number of mCs, and transforms cell types within the bristle organ (cf. Ch. 2) [997]. Neuroblast commitment in the embryonic CNS was already known to depend on the doses of Notchpathway genes [1000, 4467] when, in 1991, two labs reported that N dosage can bias SOP fates in discs. Garc´ıaBellido’s team induced marked +/+ clones in Nnull /+ vs. +/+ ﬂies and found more +/+ bristles in the +/+ controls (N dosage of clone:background = 2:2) than in the Nnull /+ ﬂies (2:1) [996]. In other words, the more N a cell has relative to its neighbors, the less likely it is

IMAGINAL DISCS

to become a SOP [112, 1458, 4193]. This same conclusion was reached by Heitzler and Simpson: in N mosaics, the bristles typically develop from cells having less N wherever tissues of unequal dose (1:2 or 2:3) confront one another at a genotype boundary [1797]. In Dl mosaics, the border bristles tend to come from cells with more Dl (4 vs. 3 doses) [1797], while in Su(H) mosaics, they tend to arise from cells with less Su(H) (2:4 or 3:4) [3820]. In summary, PNC cells compete to become SOPs, and the outcome can be affected by virtually any component in the ASC-Notch feedback loop. The dosage sensitivity of the SOP decision may help explain an odd class of dominant N alleles called Abruptex (N Ax ) [997, 1274, 3435, 3436]. Like artiﬁcially expressed N-intra, the N Ax mutations eliminate bristles by stiﬂing SOP initiation [197, 1798, 2627, 3235, 3863]. Unlike N-intra, however, their effects are ligand dependent: N Ax phenotypes are suppressed by DlLOF [992, 4780] or lower Dl dosage [460, 1798]. N Ax mutations localize to EGF-like repeats 24-29 in N’s extracellular domain [1747, 2182], while N-Dl binding relies on repeats 11 and 12 (Fig. 2.3) [985, 3544]. Nevertheless, for some reason, NAx proteins have less afﬁnity for Dl [2543]. Attempts to solve this “Abruptex Paradox” have invoked effects on N-N oligomerization [2182] or cis N-Dl interactions on the same cell [1798], but the only certain culprit is Fringe, an auxiliary ligand [992, 2543]. Fringe normally inhibits N’s response to Serrate (cf. Ch. 6), but N Ax mutations enable Fringe to activate the pathway [990, 994, 2096], possibly by hypersensitizing N to Dl [3245]. The ways in which N Ax traits are modiﬁable by downstream genes (e.g., H or gro [992]) or by AS-C dosage [996] suggest a GOF effect of N Ax on the pathway, as does the ability of excess N to mimic N Ax traits [3235, 3863]. However, NAx cannot mimic N’s ability to bypass shibire LOF [3863], and certain N Ax alleles must be LOFs based on their enhancement over a deﬁciency [460]. Surprisingly, the latter N Ax alleles do not act via Su(H), but rather via the Wingless pathway [461]. This cross-talk (which precedes PNCs) may involve binding between N and Dishevelled – a transducer for Wingless [151, 356, 1054]. Dl appears to be the sole ligand for N in setting SOP fates in PNCs because Dl null somatic clones exhibit a maximal tuft phenotype [4859]. Thus, Serrate is not an auxiliary ligand here, unlike its redundant role inside the 5-cell bristle organ (cf. Ch. 2).

Extra SOPs could be inhibited by contact or diffusion (or both) PNCs that produce a single MC sometimes contain 2 or 3 SOP-like cells, as detected by low-level expression of a neural lacZ reporter [1925]. The implication is

CHAPTER THREE. BRISTLE PATTERNS

that these “pre-SOPs” are competing [918, 1458]. Presumably, one eventually wins and proceeds to form the bristle, while the other loses and regresses to an epidermal state [918]. Curiously, some pre-SOP rivals are nonadjacent (Fig. 4c of [1925]). How can they compete via a Dl-N mechanism if they do not touch? This ﬁnding raises a key question. If Dl alone transmits inhibitory signals in PNCs, then inhibition should require direct contact, unless Dl can diffuse (see below). Given how epidermal cells are packed [4641], a SOP should touch no more than 5 or 6 cells [3641]. Until 1991 (when Ac and Sc were seen in situ), the number of PNC cells per MC site was thought to be in this range (based on the number of bristles per tuft in partial-LOF N and Dl mutants [3641, 3950] though shi LOF is more extreme [3425]), but then it was found that a mature PNC actually contains 20–30 cells [912, 3982] – far too many for an ordinary cell to reach [1458, 3272]. Two possible ways out of this dilemma have been proposed: 1. The only PNC cells that are truly in contention to become a SOP are those that express Ac and Sc at a higher level [918, 1458, 3022]. The number of such cells per PNC is indeed in the 5 or 6 range [912, 913, 3982]. In PNCs that yield two MCs (e.g., the dorsocentrals), the posterior SOP arises before the anterior SOP, and the PNC area that stains intensely for Ac and Sc appears to shift accordingly (Fig. 3.3). “Heterochronic pairs” [1925] are governed by single AS-C enhancers [1538]. The timing mechanism is unknown, but it too may rely on the AS-C [1925] because less Ac can cause the dorsocentral PNC to yield a single bristle that occupies an intermediate position [4095], and other odd spatial and temporal shifts affect SOPs when AS-C function is derepressed [1926]. 2. The SOP inhibits other PNC cells via ﬁlopodial extensions of its cell body [1802, 1810, 1813]. Filopodia indeed extend from SOPs before they divide [2387, 3630], and the ﬁlopodia of neighboring SOPs on the notum can touch (F. Roegiers, pers. comm.). Anti-Dl antibodies do not detect such extensions in situ [2296, 3270], but their visibility may be limited by their size. Intriguingly, when Dl- and N-expressing cells are cocultured in vitro, ﬁlopodia sprout from the former but not the latter: We consistently noted a morphological difference between Delta+ and Notch+ cells in mixed aggregates that were incubated overnight. Delta+ cells often had long extensions that completely surrounded adjacent Notch+ cells, while Notch+ cells were almost always rounded in appearance without noticeable cytoplasmic extensions. [1204]

53

In 1999, the old dogma of contact-mediated Dl-N signaling [2296, 3022] was challenged with the discovery of a soluble in vivo form of Dl that is snipped from fulllength protein by a protease [3479]. The protease is encoded by kuzbanian (kuz). Kuz may also cleave N [2298, 3153, 3239, 3640] ([3479] sequel), but N appears to be cleaved by a different metalloprotease [491, 2995], as well as by Presenilin [4156] and by a Furin-like convertase (cf. Fig. 2.3) [113, 712, 1723, 2589, 3335]. Somatic clones of kuz null cells exhibit bizarre phenotypes. The clone interior is devoid of bristles, but dense tufts of kuz null bristles develop at the borders wherever a MC or mC site is encountered [3640]. The phenotype can be explained if Kuz plays both a proneural and an inhibitory role [3640]. Conceivably, (1) a Kuz-dependent proneural factor “X” diffuses several cell diameters from the wild-type tissue and (2) X prompts kuz null SOPs to arise at the border, but (3) those SOPs cannot inhibit other PNC cells because they cannot make a Kuzdependent inhibitor (Dl). Assuming that Kuz’s main duty is to liberate Dl, it is easy to see why kuz LOF mutations have no effect on cell fates inside the bristle organ (App. 3): in that case (unlike the PNC situation), Dl only needs to activate N receptors on an adjacent cell and hence would not need Kuz to help it diffuse. Certainly, Dl is needed for inhibition, but it may not be the actual diffusible signal. Indeed, the notion of diffusible Dl is hard to reconcile with what has been learned about Dl-N interactions in wing veins and ommatidia. In those tissues, Dl cannot activate N unless cells express membrane-bound Dl, which grabs N’s extracellular domain from apposed cell membranes and escorts it into the Dl-on cell via endocytosis [3271]. The idea also seems incompatible with the antagonistic properties of artiﬁcially secreted Dl constructs [4207].

Scabrous may be the diffusible SOP inhibitor If the AS-C-Notch-pathway loop were solely responsible for ensuring one SOP per PNC, then the same phenotypes should be attainable by all genes in the loop. As mentioned before, however, LOF mutations of Notchpathway genes cause denser bristles than AS-CGOF mutations. Indeed, even when AS-C genes are overexpressed using powerful promoters, no NLOF -like tufts are ever seen [918, 3628]]. Why don’t high AS-C amplitudes drive the loop to saturation and transform every PNC cell into a SOP? Conceivably, even under these extreme conditions, the SOP still emits an inhibitor that prevents SOPs from arising closer than a few cell diameters. Indeed, this assumption was axiomatic in the LI model [3641].

54

IMAGINAL DISCS

-30 hrs

Diploid

a

b

-24 -21

A NP PS P N P

Haploid

-27

A S A

P S A

A P A

-18 Time when -15 SOP appears -12 -9 A D C P P D P C A A S C

-6 -3 0 (Pupariation) +3 hrs

P S C

Proneural Cluster

c

d

Inhibitory Field

e

f

CHAPTER THREE. BRISTLE PATTERNS

The irrepressible inhibitor may be Scabrous (Sca). LOF alleles of sca cause extra bristles near normal sites [1921, 2885], and Sca has attributes (listed below) appropriate for this role [178]. Paradoxically, however, its GOF effects in the wing are opposite to what would be expected: excess Sca inhibits Notch signaling by interfering with Dl’s activation of N [2462]. Sca does not appear to be a ligand for N [186, 2461]: when phage libraries of ﬂy DNA sequences were screened for N-binding proteins, Dl, Serrate, wingless, big brain, and N were ﬁshed out, but sca was not [4823]. Nevertheless, Sca does form complexes with N [3456], and sca interacts genetically with both Dl and N [179, 437, 1921, 2885, 3490]. 1. Sca is a secreted protein (soluble glycoprotein dimer) that travels at least 3 cell diameters from its source cells (R8 photoreceptor precursors) in the eye disc [2461]. A comparable diffusion range would sufﬁce for it to act as an inhibitor for bristle SOPs. 2. Sca is ﬁrst expressed in PNCs and strongly in SOPs, after which it fades from all PNC cells, except the SOP [197, 2885]. This time course mimics Ac and Sc. Genetically, sca is downstream of the AS-C: (1) excess Ac (due to Hairy wing mutations) causes sca expression in the same (normally bare) areas as ac (e.g., posterior wing blade) [2885], and (2) sca transcription ceases in an ac− sc− background [3974]. This “AS-C sca” link is due to 4 E boxes in sca’s promoter [3974]. As AS-C levels rise in the SOP, Ac/Sc/Da should turn on sca by binding these E boxes. The “AS-C Dl” link (described earlier) should also cause upregulation of Dl, which likewise has E boxes (≥12) in its promoter [2359]. However, incipient SOPs do not express

55

more Dl than other PNC cells [2296, 3270]. This uniformity was interpreted as favoring the MI over the LI Model [3022, 3270], but this fact is moot if a separate inhibitor is operating. According to one report [1203], N fades from SOPs as they mature, and this “deafness” should allow AS-C levels in the SOP to climb without interference from its Dl-expressing neighbors. Is the SOP similarly “deaf” to its own Sca? The answer must await identiﬁcation of the Sca receptor and determination of whether it is expressed in the SOP [2461, 3456]. One remaining mystery is why the sca null phenotype is so mild. Conceivably, additional SOP inhibitors might be acting redundantly with Sca [1158, 2885]. Sca must be as important as Dl [2592, 2628] because much of the second chromosome’s naturally occurring variation in bristle number (32% for abdominal and 21% for sternopleural bristles) maps to this locus [2172, 2381], and sca contributes heavily to the modiﬁcation of bristle number in artiﬁcial selection trials [1658, 2638, 2643, 2644].

Inhibitory fields dictate the spacing intervals of microchaetes If Sca is a SOP inhibitor, then it should suppress wouldbe SOPs within a ∼3-cell radius. In a hexagonally packed epithelium, an “inhibitory ﬁeld” of this size would cover 36 cells (6, 12, and 18 cells in concentric shells) – enough to reach all 20–30 cells in an average PNC (Fig. 3.7). Regardless of how Sca acts, it is worth pondering how inhibition might work in general. If each SOP creates its own inhibitory ﬁeld, then patterns could be constructed “dot by dot.” That is, extant SOPs could be used as points of reference for nascent SOPs in the same way that a

FIGURE 3.7. Origin of macrochaete SOPs on the notum.

a, b. Heminota (minus humeri) composed of diploid (2n, a) vs. haploid (1n, b) cells should look grossly alike because ploidy does not alter body size in animal species [1190, 1191]. The chief effect of ploidy is a proportional change in cell volume [1801]. Ovals denote macrochaete PNCs, most of which arise (asynchronously) before the notal region acquires the shape shown (cf. Fig. 2.6 for acronyms and Fig. 3.3 for a realistic depiction of a PNC). Dashed lines mark when each SOP appears [1925, 3374]. This temporal sequence is essentially irrelevant to the pattern of axonal fasciculation within the disc [4431, 4432] and axonal projection within the CNS [1455] (not shown). c, d. Area where the PDC macrochaete arises (enlarged). Ploidy uncouples mechanisms that depend on cell size (inhibitory ﬁeld diameter?) from those that do not (PNC diameter?). Assuming that PNCs (dark shading) come from a disc’s global prepattern, a 1n PNC will have the same area as a 2n PNC, but should contain more cells (small hexagons) because 1n cells are ∼21% smaller in diameter (0.793 = 0.5; 1n wing cells have 40% less area [3750]). Central cell (black with white outline) is the SOP. e, f. SOPs may use a diffusible inhibitor (circle = range) to prevent excess SOPs. If smaller cells secrete less inhibitor, then 1n SOPs may not be able to inhibit outlying cells, some of which will become SOPs and set up their own ﬁelds. Indeed, 1n-2n mosaics do have extra bristles at MC sites (and higher bristle densities overall) when occupied by 1n tissue [3750, 3754]. However, there is usually only one extra bristle per site and it tends to arise anteriorly or posteriorly, but not laterally. This result favors the Lateral Inhibition Model over the Mutual Inhibition Model (Fig. 3.6; see text).

56

IMAGINAL DISCS

geometer uses a compass for triangulation [800, 4657]. This supposition leads to two predictions: Prediction 1: Prediction 2:

The inhibitory radius should depend on the size of the SOP cell. Inhibitory ﬁelds set the intervals between evenly spaced bristles.

Both expectations ﬁt the LI model, but neither of them easily ﬁts the MI model. Prediction 1 is testable by using ploidy to uncouple cell size from body size. As a rule in animal species, alterations in ploidy cause ubiquitous (proportional) changes in cell volume but do not change body size [855, 1190, 1191]. Thus, the SOPs in haploid (1n) ﬂies should be half the volume of SOPs in diploid (2n) ﬂies, but 1n PNCs should be as large as 2n PNCs because PNC dimensions should depend only on body size. Under these conditions, a 1n SOP might not be able to secrete enough inhibitor to cover its PNC, in which case multiple bristles should arise (Fig. 3.7). Indeed, extra MCs develop at the normal MC sites when those sites are occupied by 1n tissue in 1n/2n chimeras [3750]. This result is consistent with Prediction 1. Prediction 2 is supported by the observations on the spatial distribution of mCs under various conditions: 1. In the 1n/2n chimeras mentioned above, the mCs are more closely spaced in the 1n areas [3750, 3754], presumably because 1n SOPs exude less inhibitor than 2n SOPs. 2. Triploid ﬂies have larger cells and larger bristle intervals than diploid ﬂies [1801], presumably because 3n SOPs exude more inhibitor than 2n SOPs. 3. Starved ﬂies have smaller cells and smaller bristle intervals [1801, 3617]. 4. Egfr LOF mutations cause smaller cells and denser bristles [1042]. 5. On the abdominal sternites of wild-type ﬂies, bristle lengths and spacing vary, and “nearest-neighbor distance” is proportional to bristle length [804]. Tergites exhibit a similar trend [801, 2083, 2690], and bristle interval varies with bristle length among bristle rows on the second-leg basitarsus (Fig. 5.1e) [1803]. 6. Females, which are larger than males, tend to have more bristles (except MCs) on the notum [1741, 4428], wing [1741], legs [1714], tergites [801, 1357, 2690], sternites [3522, 3555], and sternopleura [3522]. 7. Sternopleurae exhibit extra bristles (within a gender) when cell number increases [1471, 4036].

The above trends argue that mCs are spaced by a “bottom-up” process (“ ” denotes “causes”) [862]: inhibitory ﬁeld radius SOP size next bristle pattern.

distance to

In contrast, MCs appear to be patterned by a “top-down” strategy: Disc-wide coordinate system(?) pattern of SOPs.

pattern of PNCs

Here, we encounter a “MC vs. mC Paradox.” How can MCs and mCs be patterned differently if they each come from the same sort of PNC? The answer is: They don’t. In fact, mCs tend to come from proneural areas that are larger than the PNCs for MCs (see below) [1453], and the locations of the SOPs within those areas are not preset [913, 3954]. This indeterminacy explains the variability of mC numbers and positions (1) from ﬂy to ﬂy [3522], (2) on the two sides of the same ﬂy [432, 850, 1714, 3553, 3554], and (3) from one row to the next [1808, 1813, 1883]. In contrast, MCs are notoriously constant in pattern [3206, 3966]. Our insights into why MC and mC patterns behave differently gelled in the 1990s, but Curt Stern guessed the answer in 1956 from his studies of Minute LOF mosaics. As mentioned in Ch. 2, Minutes are genes whose LOF alleles cause small bristles [118, 2561]. Stern found several mosaics where a “not-Minute” (+/+) clone formed an extra MC near a “Minute” (Minute LOF /+) dorsocentral MC [4097] (cf. his Fig. 4). His interpretation resembles the argument above for 1n/2n chimeras insofar as he attributes the duplicate MCs to inadequate inhibition. In this same passage, he explains how asynchrony – if the hiatus is long enough – could permit the inhibition from one SOP to fade before a new bristle is initiated at virtually the same site (italics are author’s): All cases of duplicate or supernumerary bristles in mosaics may be interpreted by the assumptions (1) that the prepattern deﬁnes not sharp points but larger regions characterized by gradients in strength, (2) that the differentiation of a bristle tends to inhibit the differentiation of other bristles in its surroundings and (3) that under equal circumstances the inhibiting effect of a differentiating large not-Minute bristle is stronger than that of a Minute bristle. Therefore, if a Minute bristle develops in a mosaic and the more competent notMinute tissue covers nearby parts of the prepatterned region, then the inhibition exerted by the differentiating Minute bristle is insufficient to suppress the formation of a twin or supernumerary bristle in the [clone]. . . . [But] difficulties remain. Inhibition of bristles should be a mutual phenomenon. Why then, when it is assumed that a Minute bristle is not strong enough

CHAPTER THREE. BRISTLE PATTERNS

to inhibit a not-Minute twin, should not a not-Minute bristle inhibit the formation of its Minute twin? One might speculate that twin bristles arise when the Minute partner happens to begin differentiation earlier than the not-Minute duplicate and once having started on its developmental path is not subject to inhibition any more.

A similar rule governs the PNCs at MC sites where two SOPs normally develop (e.g., the ADC and PDC bristles within the dorsocentral PNC). Such SOPs are called “heterochronic pairs” because one member always arises before the other. Huang et al. argued that the interval between the two MCs is ﬁxed by the radius of the inhibitory ﬁeld that emanates from the “ﬁrst-born” SOP [1925]: In each case, the late SOP of the pair appears 3--4 cells away from the early one. The relief from inhibition of the second SOP could simply result from the intercalary growth of the disc, leading to an extension of AS-C expression beyond the radius of inhibition. . . . If our interpretation of the heterochronic pair is correct, it indicates that, in the notum and wing veins, lateral inhibition plays a dual role in pattern formation: first, ensuring that only one SOP will form at each site; second, allowing late SOPs to form at regular distances from early ones.

The “halo” of lower Ac-Sc expression around incipient SOPs (Fig. 3.3) ﬁts this scenario, because it implies an inhibitory inﬂuence from the SOP [912], although it could be an artifact of changes in cell size and nuclear position [1458]. ADC-PDC idiosyncrasies in ac1 mutants had long ago intimated an interdependence of paired SOPs: The two central bristles [ADC, PDC] together form an interdependent system. If the PDC develops, the ADC is also always present, but if the PDC is absent the ADC may or may not develop. Another property links the two central bristles together. Their positions on the thorax, as measured by the distance from some rather fixed landmarks, are variable in a peculiar way. When both bristles occur, the ADC is about where it would be in normal flies, but the PDC arises much closer to the ADC than a normal PDC would. When the PDC is absent, then the ADC does not form at its accustomed place but appears farther back. The apparent shift in position toward the posterior is clear-cut, though usually not extreme. [4096]

In summary, the PNCs at MC sites can contain more than one SOP (either naturally or under abnormal circumstances). In those cases, the interbristle distance appears to be set by the radius of an inhibitory ﬁeld that is produced by whichever member of the SOP pair arises ﬁrst. The use of an inhibitory ﬁeld in this way by a MC is unusual, but it is commonplace for mCs. The

57

histological evidence for these conclusions is reviewed below.

Microchaetes come from proneural stripes, not spots Notal mCs (and indeed most small bristles) develop after pupariation, whereas most MCs originate during the 3rd instar. For this reason, the same area can be patterned twice by separate mechanisms with no interference whatsoever [2427, 3067]. This “Heterochronic Superposition Trick” is used not only for MCs vs. mCs on the notum but also for (1) chemo- vs. mechanosensory (early vs. late) bristles on the legs [1803, 3628] and (2) photoreceptors vs. bristles (early vs. late) in the eyes [602, 603]. Between the stages of MC (early) and mC (late) patterning, the intervals between MC SOPs increase due to cell proliferation [1363, 3951] and epithelial stretching (during evagination). Therefore, the inter-SOP distances for MCs and mCs may not be as different at the outset as they appear in the adult. If so, then the radii of the inhibitory ﬁelds used by these different sets of SOPs might also be comparable. The inhibitory ﬁelds of the MC SOPs probably vanish before the patterning of mC SOPs begins [1458]. During the “classical” era of bristle-pattern research from ca 1954 (Stern’s work on ac1 ) to 1985 (Modolell’s cloning of the AS-C), most of the theorizing revolved around MCs, and this trend continued through 1991 when MC PNCs [912, 3982] and SOPs [1925] were documented. By that point, a dogma had emerged whose tenets were as follows: 1. Every bristle has its own PNC (2-SOP nests are exceptions). 2. AS-C action (conferring competence) always precedes Notch-pathway action (ensuring one SOP per PNC). In 1993 and 1997, analogous studies were made on SOPs [4428] and PNCs [3270] of notal mCs and leg bristles [3193]. These bristles, which are aligned in rows, arise differently from most MCs, although they share some features with 2-SOP MC nests. The heretical ﬁndings are listed below and summarized in Figs. 3.8 and 3.9 (cf. Fig. 3.7). 1. SOPs within a notal mC row do not originate in a ﬁxed sequence from ﬂy to ﬂy. Rather, late SOPs emerge in the largest gaps between SOPs that happen to develop ﬁrst [4428], just as Wigglesworth observed in

58

IMAGINAL DISCS

a

(N) (Dl)

54321

Dl N Dl N Dl N Dl N Dl (N)

(Dl)

(N)

(Dl)

(N)

(Dl)

(N)

(Dl)

(N)

(N) (Dl)

Dl & N Stripes appear (0-12 h AP)

b

Stripes h AP 1&5 0 3? 6-8 2 & 4? 8-12

Proneural Stripe (N) (Dl)

Inhibitory Field

Ac Ac Ac Ac Ac Dl N Dl N Dl N Dl N Dl (N)

(Dl)

(N)

(Dl)

(N)

(Dl)

(N)

(Dl)

(N)

(N) (Dl)

Ac Stripes & SOPs appear (8-12 h AP)

SOP

c

SOPs

h AP

5 1&3 2&4

8 10 12

d e

Late SOPs arise (12-20 h AP)

f

Ac

Dl

LOF

CHAPTER THREE. BRISTLE PATTERNS

hemipterans [4657]. These vagaries contradict any notion of prepatterned PNCs or predestined SOPs. 2. Unlike MCs, where Ac is expressed in PNC “spots,” notal and leg bristle rows are preceded by Ac stripes [3193, 3270]. The stripes could be chains of fused spots [3951], but other evidence argues that the distinction is real [1804, 3193, 4428]. 3. In the notal mC region, Dl and N are expressed in alternating stripes ∼8 h before Achaete is detectable [3270]. This “backward” sequence rules out an “AS-C Dl” link, and this conclusion was conﬁrmed when Dl stripes were shown to develop normally in an ac− sc− background. 4. Each Dl or N stripe is 3–5 cells wide [3270]. (Dl and N are both expressed outside their own stripes at low levels.) If Dl-N contests (Fig. 3.6) only occur between adjacent cells, then Dl and N must be using some other mechanism to form alternating stripes that are more than one cell wide. Hints of “proneural stripes” had previously come from heat-sensitive Nts1 and shits1 hypomorphs. Their notal rows 1 and 5 swell with extra bristles to stripe-like widths when pulsed at 6–12 h (Nts1 ) or 14–20 h (shits1 ) after pupariation [1742, 3863, 3891]. The NGOF allele N B suppresses all rows except row 1 [102, 2626, 2627]. Somatic clones that are null for all 7 bHLH E(spl)-C genes cause simi-

59

lar stripes of densely packed mCs [1794]. If each mC had its own PNC, then the LOF defects should have been punctate clumps (cf. Brd GOF ﬂies [2499, 2500]), rather than uniformly dense bands of bristles. The fact that Dl precedes Ac in stripes 1–5 suggests a “Dl AS-C” link (cf. proneural roles for N [460, 2540]), but the old “Dl AS-C” rule must still hold because (1) reducing Dl function during this period upregulates ac, and (2) raising N function suppresses ac [3270]. The “Dl AS-C” effect may initially be blocked within Dl stripes because Dl is seen ﬁrst on cell surfaces and only later in cytoplasmic vesicles (as is N). Derepression of ac in a Dl LOF background causes Ac to ﬁll the row 1–5 area (Fig. 3.8f) [3270]. Evidently, Ac stripes in wild-type ﬂies arise by a geometric subtraction. (The term “antineural” denotes inhibition of SOP initiation [1440, 2293, 4387, 4898].) 1 large Ac proneural area (Fig. 3.8f) minus 4 high-N antineural stripes (Fig. 3.8b) equals 5 Ac proneural stripes (Fig. 3.8c) Extra bristles in Nts1 ﬂies are mostly within the rows, which makes sense because an overall drop in N function should affect low-N (high-Dl) stripes ﬁrst, where

FIGURE 3.8. Origin of microchaete SOPs on the notum.

a. Heminotum (minus humerus), showing proneural stripes where Achaete (Ac) protein (c) foreshadows the 5 medial rows of microchaetes (e). Stripes 1 and 5 converge and fuse at their posterior tips (not shown), and other stripes develop laterally. Stripe 5 overlaps the ADC and PDC macrochaete sites, whose SOPs arise ≥12 h before pupariation (Fig. 3.7). These SOPs may still exude inhibition because no microchaetes form between them. b. Enlarged swath, showing cells (hexagons). Delta (Dl) protein appears at ∼0 h after pupariation (AP), is expressed in all 5 stripes (shaded) by 12 h AP (cf. table), and is detectable at low levels (“(Dl)”) elsewhere. Notch (N) protein is also ubiquitous but more intense (hatched) between Dl stripes. Whether these stripes arise via the kinds of AS-C cis-enhancers that govern macrochaetes is unknown [1538]. Also unknown is whether the early stripes constrain the later ones [3966]. c. By ∼8 h AP, Ac is congruent with Dl but is conﬁned to incipient SOPs (black cells outlined in white) by 12 h AP. SOPs appear in stripe 5, then 1 and 3, then 2 and 4 (cf. table). Inside each stripe, SOPs arise asynchronously. d. Most SOPs are evident by 12 h AP, but some emerge as late as 20 h AP. By that time, the earlier SOPs have not formed bristles as shown here, but have divided and appear as 4-cell tetrads. Late SOPs arise in the largest spaces between extant SOPs, as Wigglesworth found in hemipterans. He imagined that SOPs stiﬂe would-be SOPs nearby by emitting an inhibitor [4660] or absorbing an inducer [4657]. Only when a competent (Ac-expressing) cell ﬁnds itself outside inhibitory ﬁelds can it become a SOP. These rules produce a roughly even spacing of bristles in the available area. Proneural stripes will yield zigzag bristle rows if the ﬁelds are as wide (3–5 cells) as the stripes. Depending on how the signal is transmitted, inhibitory ﬁelds need not be circular. Here they are drawn as ovals (c, d). e. Heminotum, showing all bristles. f. Effect of lowering Dl function on Ac expression. When t.s. Dl LOF mutants are heat-pulsed at 5–10 h AP, Ac is expressed ubiquitously except along the midline (the same band that remains bare in N LOF and shi LOF mutants [1742, 3863]). The implication is that Dl normally represses ac between the “Dl stripes.” Apparently, the interstripes default to an ac-on state in the absence of N signaling. This diagram is based on data from [3270, 4428]. N.B.: It makes sense that bristles would be positioned late in development (after proliferation has ceased) because any pattern that is created earlier would be “messed up” by scattered mitoses [695]. This system thus obeys the general rule that proliferation and differentiation are mutually exclusive in eukaryotic cells [4882].

60

IMAGINAL DISCS

h

a

h

h

h

h

b

h Stripes appear (~0 h AP)

En

V

V

posterior

D

D

4 h Ac

3 2 Ac h Ac

V 1 8 Ac h Ac

anterior

7 6 Ac h Ac

D 5 Ac h

Proneural Stripe SOP

Ac Stripes appear (5-6 h AP)

c

V

4 3 21

V

En

V

D

D

V

V

V

d Fine-tuning occurs (12-24 h AP)

e

f

Ac

Ac

N is the limiting factor [1742, 3270]. Likewise, reducing Dl function derepresses ac in the low-Dl (high-N) stripes where Dl is the limiting factor, but there appears to be only minimal Ac intensiﬁcation in the high-Dl stripes [3270].

Ac

Ac

Ac

h

LOF

A priori, one would expect a signal-receiver system to be silenced to the same extent, whether the signal or receiver is reduced [184]. Clearly, however, the Notch pathway is not muzzled as much in high-N–low-Dl as high-Dl–low-N stripes: the former suppress ac, whereas

CHAPTER THREE. BRISTLE PATTERNS

61

FIGURE 3.9. Origin of bristle SOPs on the leg.

a. Distal half of a left second leg (mid-tibia to claws), showing stripes of Hairy protein that appear at pupariation [665, 3193]. Only 3 of the 4 stripes are visible from this posterior view. Transverse stripes are omitted. Dorsal (D) and ventral (V) midlines are marked. At this stage, leg segments are actually shorter and wider than drawn here. b. Entire 360◦ surface of an enlarged swath (ﬁlleted at D midline), showing cells (hexagons) and all 4 Hairy stripes (hatched). Black bar (below) shows the extent of Engrailed (En) expression, which characterizes the posterior compartment. c. Same swath, showing 8 stripes of Achaete (Ac) protein (shaded) that all appear at 5–6 h AP (after pupariation) [3193]. These proneural stripes (each 3–4 cells wide) foreshadow the tarsal bristle rows (e). The number of bristles per row (SOPs per stripe) decreases with distance from the V midline [1714]. Positions of SOPs (black cells outlined in white) are hypothetical. Their emergence has not been examined histologically. Lineage studies indicate that SOPs in stripe 1 (at least) originate from ≥2 columns of cells because row 1 bristles can come from both compartments [1800, 2449]. SOPs in stripe 1 are depicted posterior (6 at left) or anterior (4 at right) to the boundary (white zigzag line). Inhibitory ﬁelds around SOPs are omitted. d. Inferred movements (white arrows). Lineage studies suggest that (1) bracts and bristles in each row come from separate columns of cells and (2) bristle cells move ventrally (V arrows; stripes 2–7) or dorsally (D arrows; stripes 1 and 8) to one edge of their stripe [1800, 2449]. Adjustments of this kind (previously seen with scale precursor cells on moth wings [3051]) would explain why leg bristles are more aligned than notal bristles. Other “ﬁne-tuning” shifts may occur vertically, based on abnormal spacing in mutants with misoriented bristles [1810]. The horizontal and vertical shifts have been combined into one vector per SOP, although they probably happen at different times [1803] (Fig. 3.11). e. Adult left leg (posterior view), showing bristle rows 1–4. f. Effect of eliminating hfunction on Ac: Ac now appears in 4 broad stripes, implying that the 8 Ac stripes arise by repression of ac within Hairy stripes. This “subtractive” logic is similar to the strategy used on the notum (cf. Fig. 3.8). Dl and N protein distributions have not yet been assessed for the legs. Notch signaling might be repressing ac in the other 4 interstripes.

the latter do not. The reason could reside in (1) subtleties of Dl-N stoichiometry or (2) restricted distribution of other Notch-pathway components [3270]. For example, the m8 promoter drives reporter gene expression in 5 stripes per heminotum [2453], so ac could be off in high-N stripes because m8 can only be turned on there. However, Dl might be diffusing into the high-N stripes [3479] and activating the pathway there and only there. In that case, high-Dl cells would be “deaf” to their own signals [988], perhaps due to cis interactions between Dl and N molecules on their surfaces [2008].

Hairy paints ‘‘antineural’’ stripes on the legs LOF mutations in hairy (h) cause extra bristle rows on the notum [803] and ectopic mCs on the scutellum [1981, 2959], where Hairy is strongly expressed in wild-type ﬂies [665]. Extra bristles also arise on the head, wings, legs, and ﬂanks [1802, 2561]. The added bristles vanish when hLOF is combined with ac LOF , indicating an antagonism between h and ac. In fact, many of the excess bristles disappear even when ac LOF is heterozygous [2959, 4185]. This sensitivity implies a delicate balance of proneural (ac) and antineural (h) dosages in certain regions [665]. Curiously, the bHLH protein encoded by hairy does not bind Ac, Sc, or Da [68, 4452], so hairy cannot be inhibiting the AS-C via heterodimers. Hairy belongs to the same HLH subclass as E(spl)-C bHLH proteins. Like them, it should be acting as a repressor because its WRPW terminus recruits the Gro co-repressor (Fig. 2.4) [1242, 4865]. However, (1) hLOF and gro LOF phenotypes differ

[991], (2) hairy’s interaction with sc depends on a different (“Orange”) domain [976], and (3) still another Hairy domain binds a regulatory C-terminal Binding Protein [3377, 3430, 4865] (App. 5). Hairy must operate by DNA binding because it is converted from a repressor to an activator when its WRPW tip is replaced by a VP16 activator domain [2065]. Proof that it functions exclusively in this capacity was provided by two studies in 1994 [3179, 4451] that showed the following:

1. Hairy speciﬁcally binds a GGCACGCGAC sequence in the ac promoter (∼300 b.p. upstream of the transcription start site). This same consensus was ﬁshed out from a pool of random oligonucleotides by Hairy “bait,” so it is Hairy’s optimal binding site. At least two E(spl)-C bHLH proteins (M5 and M7) can also bind the core hexamer (CACGCG) with high afﬁnity. 2. In cultured cells, Hairy represses transcription of a transfected reporter gene linked to four of these sequences. The effect is abolished when the core binding site is mutated, so Hairy must exert its repression via this site. 3. In ac− sc− [4451] or wild-type [3179] ﬂies, an ac+ transgene (with its own promoter) is repressed by the endogenous h+ but is derepressed (as assayed by extra bristles) when h+ is replaced by hLOF . This result argues that no redundant gene (besides h) represses ac via this site (despite the ability of E(spl)-C proteins to bind there in vitro). The same phenotype is obtained when the core binding site is mutated,

62

IMAGINAL DISCS

so all hairy’s repressive effects must be mediated by this cis-silencer. On the legs, Hairy stripes play the same antineural role as Notch-pathway stripes on the notum. They appear before ac turns on (Fig. 3.9b), and their removal derepresses ac (Fig. 3.9f) [665, 3193]. Unlike the notum, however, there are only half as many antineural stripes relative to proneural stripes (4 vs. 8). How is ac repressed in the four other interstripes? The Notch pathway must be involved somehow because heat pulses to Nts1 ﬂies cause extra bristles between bristle rows as well as within them [1802, 1803]. Single Ac stripes on the notum and legs can produce more than a dozen SOPs without any obvious fragmentation of the stripes into proneural “clusters” sensu stricto. Hence, MC PNCs must be special cases of a more general regional identity that has been called the proneural “ﬁeld” [913, 1804, 2890, 3127, 3193]. Proneural ﬁelds can assume various shapes and yield various numbers of SOPs. Both sorts of variations are seen to a limited extent among PNCs themselves [912]. Field shapes include not only irregular spots (PNCs) and smooth stripes (leg zones), but also the expansive rectangles that Wigglesworth had envisioned for the tergite epidermis 60 years ago [4657]. Is it necessary to visualize the prepattern only as a series of constantly located, sharply defined peaks rising from a flattened valley floor? Could not rounded domes, ridges or even plateaus be other forms in the prepattern landscape? If so, then the inhibition . . . in the neighborhood of a developing structure may conceivably be less extensive than the particular boundary imposed by the prepattern, and more than one and perhaps many structures might appear in a single element of the prepattern. The arrangement of structures within each of these regions could then be controlled by the mechanism envisaged in Wigglesworth’s . . . model. [800].

In light of what has been learned about notal and leg bristles, the old MC-based models can now be reassessed. Just as haploid tissue makes extra bristles at MC sites (Fig. 3.7), it also makes more bristles per row [3750, 3754], so the number of SOPs per stripe is probably dictated by how many inhibitory ﬁelds can ﬁt therein. In wild-type ﬂies, the inhibitory ﬁeld would cover any PNC wherein a single SOP arises, whereas inhibitory ﬁelds would be dwarfed by proneural areas that foster multiple SOPs. Within the latter areas, any synchrony of SOP inception could lead to SOPs developing too close together. Thus, asynchrony may have evolved to guarantee a certain minimum spacing interval (by forcing later SOPs to arise outside inhibitory ﬁelds of earlier

ones). That interval would precisely equal the inhibitory radius wherever SOPs develop in linear sequence [805], but only the eye appears to use a trick of this sort in the creation of its photoreceptor array (cf. Fig. 7.7) [1804, 1808]. Apparently, the Notch pathway has the same duty inside as outside the proneural notal stripes (and in proneural ﬁelds generally) – to downregulate the AS-C. Dl is probably not an SOP inhibitor here (as conjectured for MCs) because it is not elevated in mC SOPs [3270]. Notch signaling must be low enough (in the high-Dl stripes) that the AS-C can spark SOPs, which then escape N-dependent inhibition and emit a N-independent inhibitor (Scabrous?) [3270]. This working hypothesis is an amalgam of the MI and LI models in which mutual inhibition sets the stage for lateral inhibition.

Leg bristles use extra fine-tuning tricks Leg bristles are intriguing because of their precise alignment and spacing. Moreover, they are organized into diverse subpatterns, unlike the homogeneous notal mCs. The basitarsi are especially rich in modular motifs [1714], and their development has been analyzed genetically [1799, 1802, 1807, 1808, 4346]. Their features are illustrated in Fig. 3.10. The 2nd-leg basitarsus has 8 bristle rows symmetrically disposed about the dorsal-ventral plane. The number of bristles per row varies from ﬂy to ﬂy, and adjacent rows vary independently [1813, 1883]. Bristle length and interval increase with distance from the ventral midline (cf. Fig. 5.1e) [1801, 1803], implying that dorsal SOPs have larger inhibitory ﬁelds. Higher ventral density may be adaptive for ﬁner resolution in detecting stimuli where the leg grasps objects (like the human palm). The 8-row trait has been retained in the genus over a 3-fold span of basitarsal circumference [64, 4120], implying that it is under disc-wide (vs. local) control, and this conclusion is afﬁrmed by its invariance in starved ﬂies [1801]. Superimposed upon the 8 rows are 5 chemosensory bristles, whose inter-row sites are also symmetric [2544]. A sixth bristle arises in the “vacancy” (distally between rows 2 and 3) in ∼30% of legs [1802]. The 1st- and 3rd-leg basitarsi have transverse rows of pale bristles on the front or rear face, respectively. These “t-rows” are used as brushes where the legs contact the eyes (1st leg) or wings (3rd leg) during the cleaning ritual [3376, 4225, 4462]. The 2nd and 3rd legs are not dimorphic, but the male 1st leg has a “sex comb” that females lack. Because the 2nd leg is simplest, its pattern may be most primitive (cf. Ch. 8) [1807, 1883, 4095]. If so, then evolution may have cobbled together the 1st- and 3rd-leg

CHAPTER THREE. BRISTLE PATTERNS

basitarsal patterns via the following “Basitarsal Elaboration Scenario” (Fig. 3.10), starting with the basic array of 8 longitudinal rows. Figuring out how genes were rewired to make such patterns might lead to clues about how evolution tinkers with development [1361]. The following steps are largely speculative (as is the rest of this section), and readers may safely skip to the next section: 1. Evolution inserted t-rows into 1st legs (between rows 7 and 8) and 3rd legs (between rows 1 and 2). T-rows are in the anterior lineage compartment on 1st legs, but they are in the posterior compartment on 3rd legs [4076]. First legs apparently kept row 7, but 3rd legs widened their t-rows to replace row 2 [4349]. N.B.: Homeotic mutations that transform A regions of 3rd legs to resemble A regions of 1st legs fail to affect P rows [1713, 4349], thus creating legs that have two symmetric sets of t-rows. 2. Evolution suppressed part of row 1 on 3rd legs. On the 3rd leg, row 1 seems to have lost its middle bristles at the compartment boundary. The proximal few bristles and the distalmost “orphan” bristle (all of which tend to arise anterior to the A/P line [1800]) persisted, but the orphan bristle moved across the midline to abut row 8, while the ventral campaniform sensillum made a similar move (cf. misplaced sensilla in sple1 legs [1810]; Fig. 5.12g). N.B.: In Ubx bxLOF ﬂies, the A region of the 3rd leg mimics the A region of the 2nd leg: the long-lost middle bristles reappear and the orphan bristle moves back to join a restored row 1 (cf. Fig. 3c of [2449]). 3. The sex comb originated as a modiﬁed t-row. Before this step, the 1st legs of both sexes probably had the modern female-type pattern (cf. D. ananassae [4095]), wherein the proximal ends of rows 1 and 8 converge, and the second bristle in row 8 mimics the corresponding bristle of row 1 (cf. the big second and third bristles on the 3rd leg). Various types of sex combs exist in the genus [1361, 4095]. Some are merely t-rows whose bristles are thick, blunt, dark, and curved. In others, the combs are oriented longitudinally. Cell lineage studies in D. melanogaster show that its comb begins as a distal t-row, which then turns ∼90◦ (Fig. 3.11g–i) [4344]. The following conjectures pertain mainly to how the sex comb rotates during development, but they also have implications about how it evolved: a. The comb has more bristles than any t-row, but distal rows are also broader than proximal ones,

63

b.

c.

d.

e.

so a growth gradient may exist along the segment. Indeed, the comb probably rotates via growth [3446], rather than by cell migration [1357]. The rotation may push the t-row just above it since this row often bends away dorsally. When ﬂies carrying a t.s. sex-transforming mutation are shifted to high temperature, the sequence of their comb defects reveals that bristle number is set before bristle type [281]. One t-row seems to be suppressed to make room for the rotation [2978, 2979, 3566, 3987, 4344], although two t-rows might be merging to form a single comb [4109]. Intersexual basitarsi may reveal a “missing link” transitional stage [1845]. Cell death may be involved in the rotation because this region hypertrophies (an overreaction to expanded apoptosis? [3442]) in cell-lethal mutants [3442, 3964]. The “central bristle” [1714] seems homologous to the 2nd leg’s distalmost row-8 bristle. It probably belonged to the sex comb but detached and migrated to its central site in the bare triangular area. Similar detachments of row 8’s middle bristles from the t-rows may have occurred that led to their elimination (cf. the male vs. female patterns) [4344].

If this evolutionary scenario is even partly correct, then it connotes a thorough reprogramming of cell behaviors. We know virtually nothing about the genetics of those alterations. In contrast, we are beginning to understand how SOPs rearrange within incipient bristle rows (Fig. 3.11). By heat-shocking wild-type pupae, abnormal phenotypes can be induced [1803], and the sequence of their sensitive periods suggests the following stages: 1. Lateral movements reﬁne SOP alignment. One odd phenotype in h null legs is bristle misalignment [3193]. Conceivably, SOPs on wild-type legs use Hairy/Ac boundaries as guidelines for alignment. If so, then the loss of these lines in h null legs might be causing the disorder [3193]. Additional evidence for transverse SOP movements comes from cell lineage: in certain rows, the bristle SOPs arise ventral to their prospective bract cells, while in others the bristles arise relatively dorsally (Fig. 3.9) [1800, 2449]. Because each bristle eventually lines up with its bract, the SOPs must undergo directed lateral movements within each proneural stripe (Fig. 3.11a–c). Stripes 1, 2, 4, 5, 7, and 8 lack a Hairy/Ac boundary at the edge that their

64

IMAGINAL DISCS

2nd leg D

3rd leg

P

4 3 2

V 1

A 8

7

D

D

6 5

P

4 3

V

(2)

1

A 8

7

D 6 5

100 µm

evolved?

evolved? transverse rows orphan bristle

1st leg: female D

P

4 3 2

V 1 8

1st leg: male A 7 6

D

D

5

P

4 3 2

V 1 8

A 7 6

D 5

evolved?

sex comb central bristle transverse rows

CHAPTER THREE. BRISTLE PATTERNS

SOPs supposedly seek. It is thus unclear what “homing” cues these SOPs use. 2. Proximal-distal movements reﬁne SOP spacing. Although no SOP movements have yet been witnessed in ﬂies, scale precursor cells have been shown to rearrange on moth wings [3051]. Those shifts appear to be mediated by ﬁlopodial extensions from the precursor cells, and the same ﬁlopodia may help the cells within each scale row acquire their regular spacing. Evidence for a ﬁlopodium-mediated spacing mechanism in Drosophila comes from phenotypes of mutants that have abnormally oriented bristles. In the wild-type, each row’s bristles face in the same direction. When mutant bristles face one another, they tend to have an unusually large space between them, while bristles that point away from one another tend to have an unusually small space. These correlations are explicable if SOPs are repelling one another using ﬁlopodia that are longer on one side than the other (Fig. 3.11d–f) [1810]. Such ﬁlopodia have indeed been found [2387, 3630]. Short-range adjustments of these kinds could explain why bristle rows are neater on the legs than on the notum [805, 1813, 4428]. Mutant legs that fail to elongate fully

65

have bristle rows that are as jagged as notal rows [4348], so ﬁne-tuning may require proper evagination. Notal SOPs probably differentiate in situ (i.e., wherever they arise within Ac stripes), without any subsequent corrective movements to reduce the scatter. Leg SOPs might become mobile after delaminating [1745] since they (or their descendants) could then crawl freely on the underside of the epidermis without needing to displace other cells within the crowded monolayer. Consistent with this reasoning, marked bristles are often isolated from somatic clones induced on legs [544] and tergites [1371], but not the notum [521, 1373, 1797]. Within each t-row the bristle sockets touch one another, but adjacent t-rows are separated by several cell diameters. It is therefore unlikely that t-row SOPs use isodiametric inhibitory ﬁelds [4641]. Diffusion of the inhibitor could be channeled along one axis, but it seems more plausible to imagine that the t-rows and sex comb “self-assemble” from scattered SOPs that join into tandem ﬁles by homophilic adhesion. Homophilic adhesion could also explain the tandem bristles in the medial row of the wing margin [1741]. Some sort of cellular cooperation must be occurring in both cases because sex combs and marginal rows can reform when leg or wing discs are dissociated and reaggregated [1356, 3424, 4334].

FIGURE 3.10. The four types of basitarsal bristle patterns in D. melanogaster. Each segment is drawn as if slit along its dorsal

(D) midline and spread ﬂat (P, posterior; V, ventral; A, anterior), with its proximal edge at the top. Only the 1st leg is sexually dimorphic. The 2nd-leg pattern is simplest and maybe most primitive. Its basitarsus has 8 rows of mechanosensory bristles (numbered at top), each of which bears a bract (triangle) at its base. The 5 curved “bractless” bristles reside between the rows and are chemosensory [3061]. Also shown are sensilla campaniformia (stretch detectors [4342, 4841, 4887], white circles) and trichoid hairs (small “v”s). Between rows 1 and 8 almost every cell makes a hair, so the hair density indicates cell size on the segment. There are ∼2,000 epidermal cells on the 2nd-leg basitarsus [1801]. The 1st- and 3rd-leg basitarsi augment this basic pattern with transverse rows of lighter-colored bristles. Similar rows decorate the tibias of those legs, but most of the tibial transverse-row bristles lack bracts [1714]. Transverse rows serve as brushes for cleaning the eyes (1st leg) or wings (3rd leg), and they are ideally located (A vs. P) for this role [4462]. Bristle lengths and intervals tend to increase from V to D in all patterns. In males, the most distal transverse row rotates ∼90◦ during development to form the “sex comb” [4344], which may function during courtship [1692, 4039]. Males also differ from females in having extra bractless bristles between rows 5 and 6 [1714]. In the hypothetical scheme indicated by the arrows [1883, 4096], all three leg pairs originally resembled the 2nd-leg pattern. Transverse rows then evolved in 1st and 3rd legs of both sexes, and males acquired a sex comb. The central and orphan bristles may be vestiges of these changes (see text). Alternatively, evolution might have gone the other way (i.e., simplifying an initially complex pattern). In Polycomblike LOF mutants, the 2nd leg acquires two sets of transverse rows (not shown) [1122]: one set on its A side (plus a sex comb in males) where it becomes a 1st leg and another set on its P side where it becomes a 3rd leg (see [1713]). This “homeotic schizophrenia” is a default state (cf. Table 8.1) where the Polycomb Group of “memory genes” malfunctions, thereby allowing certain homeotic genes to stay on where they should be off [1508, 1509]. Such defaults can be equivocal, however. Indeed, an opposite type of default is seen when the latter homeotic genes are disabled [4149] – i.e., 1st and 3rd legs look like 2nd legs. Drawings are for left legs from actual wild-type ﬂies but are idealized insofar as (1) segments are not perfect cylinders, (2) hairs are not so neatly arranged, and (3) color shadings in vivo make it difﬁcult to discern where transverse rows end and longitudinal rows begin, and (4) segment widths are exaggerated to avoid bristle overlaps (lengths are accurate). See also App. 7.

66

IMAGINAL DISCS

Observation 2

Inferred cell movements

1

2

D

V

1

D

2 V

1

D

V

y

b wildLOF type sple

d

c wildLOF type sple

wildLOF type sple

1

1

1

1

2

2

2

2

3

3

a

4

4

>d

3 3

d

e

4

4

f

transverse rows

g

y sex comb

h

i

CHAPTER THREE. BRISTLE PATTERNS

The chimeric composition of the reconstituted patterns proves that physically separate (differently marked) cells can align, but it is not known whether this process requires regrowth and repatterning. Hawaiian Drosophila have a tandem ﬁle of alternating “bristle-bractbristle-bract . . . ” cells in basitarsal row 8 [1801], and this peculiar row (bracted bristles are typically separated by several ordinary epidermal cells [1598]) may have evolved via heterophilic association of bristle and bract cells.

Chemosensory leg bristles are patterned like notal macrochaetes As mentioned above, the 2nd-leg basitarsus has 8 rows of mechanosensory (MS) bristles and typically 5 chemosensory (CS) bristles between the rows (Fig. 3.10). Each MS bristle bears a bract on the proximal side of its socket, whereas CS bristles are “bractless” [1714]. In many ways, basitarsal CS bristles develop like notal MCs, while the MS bristles develop like notal mCs: 1. There is an order of magnitude fewer CS than MS bristles on the 2nd-leg basitarsus (5 vs. ∼70) [1802], analogous to the disparity between notal MCs and mCs (26 vs. ∼225) [1741]. 2. Like notal MCs [3966], the number and positions of CS bristles are constant, whereas MS bristles vary in number and position from ﬂy to ﬂy (and on the left vs. right sides of individual ﬂies) [1714, 4344].

67

3. CS bristles originate before MS bristles [1803, 3628], just as MC SOPs precede mC SOPs [1925, 4428]. Thus, patterning is essentially a two-act play on both the leg and notum: in Act I a few (CS or MC) SOPs arise in a ﬁxed array, and in Act II many more (MS or mC) SOPs arise relatively randomly inside parallel proneural zones [3954]. The ability of the later kinds of patterns to form independently of (and be superimposed upon) the earlier ones is also seen on the wing margin [353, 882, 1741] and has been proposed for the integument of other insects [2425]. 4. Under certain circumstances, both CS bristles and MCs can form rows. Although CS bristles are not arranged periodically on the basitarsus, rows of such bristles are manifest on the tarsus as a whole [1811], the tibia [149, 3141, 4481], and the wing margin [1741], and other Drosophila species have strikingly regular rows of evenly spaced CS bristles on the legs [667]. Likewise, related genera have rows of evenly spaced MCs on the notum [1354], and these rows emerge (atavistically?) in D. melanogaster when proneural potential increases [1354]. Bristle intervals in CS or MC rows are larger than in adjacent MS or mC rows, presumably because later intercalary growth pushes SOPs apart after their initial interval is created by (standard size?) inhibitory ﬁelds [1354, 2959, 3950]. 5. CS bristle sites are immune to changes in cell size or organ size (and hence appear to be part of a global

FIGURE 3.11. Movements of bristle cells inferred from various studies. Bristles are denoted by circles and bracts by triangles.

Three types of hypothetical movements are shown – none of which have actually been witnessed yet. a–c. Lateral shifts that may align SOPs in longitudinal rows [1800]. a. Sections of rows 1 and 2 from a left leg (D, dorsal; V, ventral). Wavy lines indicate the kinds of boundaries seen in gynandromorphs. Male tissue is marked with yellow LOF (y). Curiously, bristles within a row tend to be more closely related to each other than to their own (physically closer) bracts. b, c. These afﬁliations are explicable if SOPs in each proneural stripe (Fig. 3.9) migrate in one direction (dorsally for row 1, ventrally for row 2) to a different column of cells where they induce neighbors to become bracts [1800, 1808]. Shaded zones in b and c denote y+ tissue as in a (they are not Ac stripes), and triangles in b mark future bracts. d–f. Proximal-distal shifts that may ﬁne-tune SOP intervals within those rows [1803, 1810, 1813]. d. LOF mutations of spiny legs (sple) cause bristles to be misoriented along with their bracts, usually a 180◦ polarity reversal (third bristle). Strangely, intervals between bristles that face one another tend to be larger (“>d”) than wild-type distance (“d”), while spaces between bristles that face away from each other tend to be smaller (“
68

positioning system of some kind), whereas MS bristle spacing and number depend on cell size and cell number, respectively [1801]. The same is true on the notum [3966], where a prepattern seems to dictate positions for MC PNCs and mC stripes but not to “micromanage” mC SOP sites within those stripes. 6. Like notal MCs, CS bristles are more affected by sc LOF than ac LOF mutations [1802], while the opposite is true for notal mCs and MS leg bristles [1360, 1453]. For these reasons, “constellation” patterns (notal MCs and CS leg bristles) and “row” patterns (notal mCs and MS leg bristles) were thought to be fundamentally different (Fig. 3.1). It therefore came as a surprise when MCs were coaxed into a mC-like pattern by gene dose manipulations (see below), implying that both types of patterns are divergent outputs of a common mechanism.

Extramacrochaetae superimposes an uneven antineural ‘‘mask’’ The extramacrochaetae gene (emc) was isolated in a screen for mutations that interact with the AS-C in a dose-dependent way [408]: LOF mutations were sought that produce extra bristles when the ratio of the tested gene to the AS-C was 1:3 or 1:4, instead of the normal 2:2. Despite the large scale of the screen, the only lesions recovered were in emc and hairy, although Notchpathway genes have similar properties [733, 997, 2690]. Thus, all genes of this kind may now have been identiﬁed. Although emc and hairy both behave as trans-repressors of the AS-C, emc LOF alleles interact mainly with sc and affect notal MCs, whereas hLOF alleles interact mainly with ac and affect notal mCs [2959, 2960]. In other body regions their effects overlap more, and their interactions with ac and sc are less restricted [1349]. Indeed, emc’s link to MCs (whence its name) is not inherent in the protein because mCs can be removed by overexpressing emc just before their SOPs arise [4453]. Like Hairy and the E(spl)-C effectors of the Notch pathway, Emc also has an HLH motif, but it lacks a basic domain for binding DNA [1156, 1388]. This feature alone sufﬁces to explain its mode of action [213, 3083] because (1) any dimers that Emc forms with Ac, Sc, and Da will have only one basic domain, but (2) two basic domains are needed to bind DNA [286]. Indeed, Emc does block DNA binding by Ac, Sc, and Da in vitro [589, 3179, 4452, 4453], and this “inert decoy” effect can been mimicked by a Da construct whose basic domain has been deleted [1754]. Hence, Emc silences the AS-C post-translationally

IMAGINAL DISCS

by sequestering Ac, Sc, and Da in inactive heterodimers. Curiously, when a basic domain from L’sc is substituted for the corresponding piece of Emc, the chimeric protein can still block E-box binding by L’sc/Da dimers [2722]. Conceivably, the inserted domain cannot dock properly with DNA because the rest of the Emc protein has evolutionarily (through disuse?) lost the ability to orient this region at a suitable angle [2722]. By adding extra doses of sc in an emc LOF background, the number of notal MCs can be increased far above the wild-type condition. Interestingly, as new MCs arise, they form rows, as if they are now obeying patterning rules that govern mCs [2959]. Indeed, the aligned MCs shift positions as new ones are added so as to maintain even spacing, so they cannot merely be sprouting from ﬁxed “cryptic singularity” sites. How can MCs acquire organizational attributes of mCs, given that MCs and mCs come from distinct proneural ﬁelds (PNC vs. stripe)? Because some MCs reside where mC rows later develop, emc LOF mutations might just be accelerating the process of stripe formation so that it intrudes into the time window for MC SOP initiation [1926]. The following facts support this hypothesis: 1. In emcLOF mutants, two rows of ectopic MCs develop along the same dorsocentral and presutural lines [1349, 1926, 2959] where mC SOPs emerge ﬁrst [4428]. Extra MCs can also be induced in these zones by overexpressing sc ubiquitously [3628]. 2. Most MC SOPs arise earlier in emcLOF mutants than in wild-type ﬂies [1926] – indicating a general acceleration of bristle development. Indeed, the plethora of “MCs” may just be an illusion resulting from a premature initiation of SOPs. If mC shaft cells start endoreplication early enough, then they could grow to the size of MCs (cf. Ch. 2), as has been inferred for mutant tergites [2690]. 3. “Excess function” (Hairy wing) mutations in the AS-C put extra MCs along the dorsocentral line [191, 635, 1577], indicating a proneural proclivity along these corridors prior to mC SOP emergence [1926]. (See [3890] for evidence of a similar tendency on the scutellum.) 4. The overall level of Sc protein increases in emcLOF mature wing discs, and the pattern of accumulation changes [912] such that the dorsocentral PNC expands to form a vague stripe along the future anteriorposterior axis. In wild-type ﬂies that stripe does not appear until ∼8 h later [3270]. These effects, and the reduction of Sc in emc GOF discs [912], are surprising, considering that Emc is supposed to affect the AS-C

CHAPTER THREE. BRISTLE PATTERNS

post-transcriptionally, but they may be indirect (see below). Heterochronic changes of this sort may have been instrumental in bristle pattern evolution, because the emc LOF array resembles MC designs of more primitive dipterans [1354, 2959]. Emc is expressed ubiquitously [1388], so one of its functions appears to be to set a threshold which AS-C activity must exceed to evoke SOPs [637, 997, 1926, 3982] – the same sort of argument proposed earlier for the Notch pathway’s role inside PNCs (the MI model). Essentially, Emc would form an inhibitory “ocean” that lets peaks (PNCs) and ridges (stripes) of a submerged prepattern landscape rise above “sea level” at certain times to make proneural territories [918]. This metaphor is emblematic of Turing-like models that appear to be irrelevant here since they invoke diffusible molecules [1727, 2806]. However, the same rules of damped autocatalysis can govern cellular automata [1033, 1437, 2651, 2694, 4699] that may be applicable. The ocean analogy is an oversimpliﬁcation because the amount of emc RNA actually varies in a landscape of its own throughout the disc epithelium. Its density tends to be low wherever Ac and Sc are expressed [913, 4453]. Although the complementarity is only approximate, it suggests a second function for Emc: to restrict the regions where proneural ﬁelds can arise by suppressing AS-C transcription [997]. Indeed, many of the extra bristles in emc LOF mutants are located in areas that are normally bare [1349], and the AS-C is derepressed at those sites [589, 3982, 4452, 4453]. In wild-type ﬂies, emc and AS-C expression are both high in some naturally nude regions, so Emc’s antineural effect there must only be post-transcriptional [589, 913]. Heterogeneous transcription of emc inside some PNCs implies a third role (aside from its global “baseline” and “carving” duties) – to conﬁne proneural potential to a subset of cells within certain PNCs [637, 913, 2062]. In such cases, the mask (of high-level emc expression) partly overlaps the PNC, rather than skirting it. The dynamics of emc and AS-C expression inside the dorsocentral PNC are revealing in this regard: After the PDC precursor has emerged, strong emc and acsc expression co-exist in the anterior part of the cluster, an area in which the ADC precursor later appears. When this SOP arises, the emc expression has been extinguished in that area. [913]

Consistent with the idea that Emc helps “ﬁne-tune” MC sites [1458], MCs are indeed positioned less accurately

69

in emc LOF mutants than in the wild-type. Particular bristles are displaced by as much as several cell diameters [1926] – as if Emc’s absence allows the area of maximal competence to broaden and shift within the PNC. Such shifts probably also occur in mosaics wherever a PNC is bisected by the boundary separating lower from higher AS-C doses [912, 1794] because the low-dose areas would be artiﬁcially mimicking the natural role envisioned for Emc [913]. Another implication from those mosaic studies is that Emc need only reduce proneural activity by a factor of two in order to perform its spatial sharpening function. Gradients of emc transcription within certain PNCs appear steep enough to span at least a 2-fold range [913]. In summary, emc affects the AS-C in time (by damping its protein accumulation) and in space (by overlaying an uneven antineural “ﬁlter” on the epidermis). This antineural emc mask (plus the prohibitive stripes painted by N and hairy) helps explain the amazing ability of bristles to still form in normal patterns, even when AS-C genes are uncoupled from all their positionspeciﬁc enhancers [1454, 1458, 1804]. 1. When ac− sc− ﬂies carrying a hs-sc [3530, 3628] or hsase [1079] transgene are heat shocked, bristles develop mainly at normal sites, despite the proneural competence that is supposedly thereby induced throughout the epidermis. 2. Bristles still arise mostly in normal areas when these same transgenes are overexpressed in a normal (ac+ sc+ ) background [438, 1079, 3628]. 3. Similar results are obtained in both genetic backgrounds (and even if the entire AS-C is deleted) when sc or l’sc is overexpressed under Gal4-UAS control [1854]. Clearly, topographic cues outside the AS-C must be constraining proneural potential. Emc provides at least some of these cues because emc LOF (in a background where ac is derepressed) decreases the positional precision of normal MCs and increases the scatter of extra bristles in general [913]. Because emc transcription is normal in ac− sc− discs [913, 4453], its peaks and valleys must be dictated by prepattern factors at a higher echelon in the chain of command. Competence is probably also gated by mitotic quiescence [637, 4427], and the dosage of da becomes limiting when AS-C monomers supersaturate the system [438, 918, 1854]. All trans-regulators of the AS-C thus far discussed (Notch pathway, hairy, emc) exert negative control in deﬁning (or reﬁning)

70

IMAGINAL DISCS

Key

4

?

?

3

?

?

?

?

?

?

?

+

~Wild phenotype Wild genotype Missing bristles

Buffered

2 1

?

?

0 1 2 3 Doses of AS-C

4

b

0

1 2 3 Doses of ac+

3

?

?

?

?

0

1 2 3 Doses of sc+

Da

Ac

4

1

c

CtBP

PLSLV WRPW

Hairy

+ + -300 b.p.

?

?

?

Gro

Emc

Sc

+

E

-200

E

-100

ac

Buffered

ac-GOF h-LOF pyd-LOF

AH 1

PA H

0 0

+1 b.p.

AHP

2

1

Wild-type

AP HP 2

3

Genotype

time window

i

4

f

Hairy

h

E

?

CACGCG

CAGCTG

N

?

2

PLSLV WRPW

g

?

orange

e d

?

0

Phenotype

a

0

4 +

Doses of emc

Doses of hairy

Extra bristles

Less AS-C activity

More AS-C activity SOPs arise

Missing bristles

Buffered

Inhib. fields Wild-type

Buffered

Extra bristles

CHAPTER THREE. BRISTLE PATTERNS

AS-C expression. Upstream factors also exist which establish spots or stripes of proneural expression by activating the AS-C, but they tend to belong to pathways that are tailored to the needs of individual discs (see below).

Dose dependency implies that HLH proteins ‘‘compute’’ bristles When the amount of a proneural or antineural protein deviates too far from its wild-type level, the resulting pattern abnormalities can be “cured” by artiﬁcially adjusting the amount of an antagonist until the deviation is “titrated” (Fig. 3.12) [408]. (Da also has dose-dependent interactions with AS-C proteins, but they are positive [949].) Examples include 1. Higher doses of emc [1348] or h [1354] erase extra bristles caused by ac GOF mutations.

71

2. Heterozygosity for AS-C deletions erases extra bristles caused by hLOF or emc LOF or both [2960]. 3. Heterozygosity for ac LOF erases extra bristles caused by hLOF [2959, 4185]. 4. Halving the AS-C dose erases extra bristles caused by half doses of N or Dl [997, 2690]. Imbalances among pro- and antineural gene products may account for the bald patches seen in interspeciﬁc Drosophila hybrids [318, 3980, 4238, 4239]. In such ﬂies, certain SOPs emerge but then abort differentiation. Hybridization may be disturbing the relative amounts of HLH agonists vs. antagonists at various sites [3980]. Those amounts would be co-adapted within species but incompatible across species [2901]. The incompatibility presumably arose via genetic drift in the parallel lineages of ﬂies after their divergence from a common ancestor [4259].

FIGURE 3.12. How HLH proteins “compute” bristles. Proneural proteins (Ac, Sc, Da) prod cells to become SOPs, whereas antineural proteins (Hairy, Emc) restrain them. a–c. Dose-dependent interactions (see key), based on data from [408, 1348, 1354, 2959, 2960]. a. Wild-type females have two AS-Cs (one on each X), and “dosage compensation” rectiﬁes the level in males to parity by a chromatin-remodeling trick (not shown) [1633, 2183, 3760] that is poorly understood [307, 308, 3249, 3997]. The phenotype resists change when AS-C dose deviates by up to a factor of two (halved to 1 or doubled to 4), and such “buffering” is also seen with hairy+ (b) or emc+ (c). However, extra bristles arise when increases in Ac or Sc combine with decreases in Hairy or Emc. d–f. HLH dimers that affect a cell’s ability to become a SOP. Shading conforms to Fig. 2.4: basic region (black), helices (hatched), loop (gray), and remainder (“noodle”). d. Ac/Da (and Sc/Da) heterodimers stimulate transcription of ac by binding “E boxes” (“class A” sites [1242, 3179]) in the ac promoter (g): CAGCTG (−262 and −195) or CAGGTG (−58) [2722, 4452, 4453]. They also activate sc [918] (not shown). These effects are blocked by Hairy and Emc, but in different ways. e. Hairy does not dimerize with Ac, Sc, or Da [68, 4452]. Its homodimers bind upstream of ac at CACGCG (a variant “N box” or “class C” site) and repress ac transcription (g) [3179, 4451], possibly via the “orange” domain [976], but more likely by recruiting Groucho (Gro, a long-range silencer that binds WRPW) [1242, 4524, 4865], C-terminal binding protein (CtBP, a short-range quencher that binds PLSLV) [2180, 3377, 3430], or other co-repressors such as dDrap (not shown) [3648]. f. Emc sequesters Ac, Sc, and Da in dimers that are inert because Emc lacks a basic (DNA-binding) domain [589, 3179, 4452, 4453]. As a result of this “broken pliers” conﬁguration, ac and sc cannot auto- or cross-activate in Emc’s presence. Emc’s effects in vivo may be greater on sc due to when (vs. where) emc is expressed. This “disabled dimer” trick is also used in the LIM-HD and POU-HD families, where Beadex [2854, 3908] and I-POU [4380] play roles analogous to that of Emc. This process whereby an inhibitor sequesters an activator is called “squelching” [1984, 4806] to distinguish it from “quenching” (i.e., the blocking of an activator by an inhibitor after both have bound a DNA site) [1600, 1984, 2350]. g. Promoter region upstream of the ac transcription start site (crooked arrow). E and N boxes are marked; “+” = activation; “−” = repression. h. An example of buffering, where three mutations were studied singly or combined: ac GOF (A, a.k.a. Hairy wing), hairy LOF (H), and pyd LOF (P). All of them increase activity of the AS-C [635, 733, 3179, 4451]. The number of mutations per ﬂy is along the x axis; the number of extra scutellar macrochaetes is along the y axis [3069]. Phenotypes remain nearly wild-type until the double-mutant threshold (cf. a–c), whereupon synergism ensues. i. Time Window Model for pattern buffering. In each panel, the x axis is time, the upper triangle denotes gradual production of SOPs in a proneural ﬁeld, and the lower triangles denote the time needed for SOPs to extend inhibitory ﬁelds (they are not really sequential). In wild-type ﬂies (middle rectangle), the time available for patterning (window width) exceeds the time needed (upper triangle width), so slight changes in AS-C activity (2nd and 4th rectangles) may merely shift the timing of events. In contrast, more extreme changes (1st and 5th rectangles) may additionally alter the rate of accumulation of Ac and Sc (gradient slope). High-AS-C ﬂies should make SOPs so quickly that they cannot fully extend inhibitory ﬁelds before new SOPs arise nearby (leading to extra bristles), whereas low-AS-C ﬂies will make SOPs so slowly that time expires before the wild-type number is attained. Whether HLH proteins also help terminate this time window is unknown, but a HLH Hourglass has been found in vertebrates [2293, 2294] that uses proneural and antineural bHLH antagonists. See also App. 7.

72

Evolutionary drift in “volume settings” of interacting HLH genes might also explain the intersexuality of many hybrids in this genus [1174, 1882, 1883] and other insect groups [1520, 1524, 3398]. Gender-speciﬁc traits in Drosophila appear to be decided by the ratio of X chromosomes to autosomes [1173, 4077] , as measured by relative doses of gene products [3266]. Remarkably, the numerator and denominator elements include the proneural bHLH proteins Scute [1028] and Daughterless [3268] , the panneural bHLH protein Deadpan [209], and the antineural HLH protein Extramacrochaetae [1155] , suggesting that evolution may have commandeered a cassette of HLH neural-patterning genes for secondary duty in sex determination [1173, 2018, 4824]. HLH genes decide cell fates not only in PNS neurogenesis and sex determination, but also in photoreceptor initiation [182, 1076] , CNS neurogenesis [485, 3981] , myogenesis [2177] , and gliogenesis [1464] , as well as in the development of midline mesectoderm [899] , Malpighian tubule tip cells [4672] , appendage tips [1166] , tracheal tubes [4548] , salivary ducts [899] , endodermal tissues [4269], etc. [914, 1343, 1744]. Like Numb, therefore, HLH transducers function at an abstract level that transcends histotype (cf. Ch. 2). Collectively, they act like analog-to-digital transducers to ensure all-or-none outcomes [816, 2958, 3186, 4584]. In short, cells use HLH proteins to do arithmetic. The virtuosity of HLH input/output devices is illustrated by the sex-determining genes, which reliably produce discretely male or female (vs. intermediate) bristles in sex combs despite X:A ratios that range between 0.5 (male) and 1.0 (female) [4370] . The choice of bristle type is made on a cell-by-cell basis [3734, 4105] a few hours after the number of sex comb SOPs is set [281, 2106]. What kind of math is being used to convert quantitative signals into qualitative states? The “SOP Computer” seems to operate by combining positive and negative scalar inputs (Ac, Sc, Da, Emc) as HLH heterodimers. For the scheme outlined below to work, Ac and Sc must be rate limiting for dimer formation with Da and Emc. If Da/Da dimers form too readily, then Da’s presence before Ac and Sc appear [905, 4435] could be problematic (although dimer preferences could be imposed biochemically or sterically by thirdparty agents [245, 286, 2046, 3083]). Indeed, Da dimerizes only weakly with itself [588, 4453]. 1. Upstream trans-activators of ac and sc intensify at certain epidermal sites, bind site-speciﬁc enhancers near ac and sc, and start to evoke Ac and Sc at time

IMAGINAL DISCS

2.

3.

4.

5.

“t1 .” Ac and Sc form heterodimers with Da or Emc [68, 589, 4452], instead of binding each other or homodimerizing [588, 918, 1484, 3974, 4453]. In these proneural areas, the number of Ac/Da and Sc/Da heterodimers per cell will increase at a rate “r1 ” that is damped by diversion of Ac, Sc, and Da monomers into inert heterodimers with Emc [4453]. Indeed, all these heterodimers form readily [68, 589, 4452], and Emc has been shown to inhibit E-box binding [4452] and transcriptional stimulation [589, 4453] by Ac-Sc-Da combinations in a competitive, dosedependent manner. Eventually, Emc is depleted (unless Emc is resupplied via transcription), and this event marks the ﬁrst titration threshold “T1 ” [1387, 4584]. Thereafter, the rate of Ac/Da and Sc/Da accumulation will increase to “r2 ” (assuming there was initially more Da than Emc). At some point, the concentration of Ac/Da and Sc/Da heterodimers crosses a second threshold “T2 ” that triggers autocatalytic production of Ac and Sc [918] via (1) direct feedback of the heterodimers on E boxes near ac and sc [2722, 4452], and (2) indirect feedback via sens, whose promoter has E boxes and whose product binds near sc and ac [3127]. Levels of Ac and Sc rise exponentially to saturation (limited by Da) [997]. The cell becomes a SOP (via target genes whose E boxes bind Ac/Da or Sc/Da) and emits a signal that prevents nearby cells from making more Ac and Sc. SOPs continue to arise within the proneural area (in either a stochastic or a spatially biased manner) until time “t2 ,” when all competent cells have either become SOPs or are inhibited by nearby SOPs.

This algorithm does not explain why Sc transcription rises in the wing disc when Emc is reduced [912], because Emc only acts post-transcriptionally and autoregulation of AS-C loci is only supposed to occur in SOPs. Conceivably, some trans-activators of the AS-C may also be bHLH proteins that could be disabled by Emc [1387]. The only gene so far identiﬁed upstream of emc itself is polychaetoid (pyd) [4237]. Pyd’s presence in intercellular junctions suggests that emc might be activated by intercellular signaling. The HLH scheme shows how easy it is to do math with molecules by titrating monomers. It uses addition (Ac + Sc = Total proneural), subtraction (Total proneural − Emc = Net proneural), and multiplication (feedback in Step 4), but not division. Indeed, it now appears that even the famous X:A “ratio” is an illusion of a

CHAPTER THREE. BRISTLE PATTERNS

procedure that strictly relies on counting [816]. The dose sensitivities of Notch-pathway components are probably also due to dimer interactions – the most obvious of which is the docking of Dl and N in trans [1204, 3544] and possibly also in cis [2008] at cell surfaces. Titration-based arithmetic may also operate at the RNA level. A motif exists in the 3 -UTRs (untranslated regions) of transcripts from hairy, emc, the E(spl)-C genes m3, m4, m5, and mγ , and the m4-related genes Bearded, Bob (Brother of Brd = actually 3 genes A, B, and C), and Tom (Twin of m4) – all of which act antineurally [2382, 2386, 2499]. This “GY” box reads GUCUUCC (except for emc’s GUUUUCC). The complementary CAGAAGG appears in 3 -UTRs of transcripts from the proneural genes ac, l’sc, and atonal [2386]. Hence, mRNAs from these proand antineural genes could form heteroduplexes that could conceivably modulate transcription, mRNA localization, processing, turnover, or translation [233, 1219, 1702, 2686, 2920, 2965, 3400]. Docking between hairy and ac mRNAs might explain why interactions between these genes are so dose sensitive (Fig. 3.12), despite the failure of their proteins to dimerize [68, 4452]. Other motifs in these antineural-class UTRs include “Brd” (AGCUUUA) and “K” (UGUGAU) boxes, both of which decrease accumulation of transcripts [92, 2382, 2384, 2385].

Robustness of patterning may be due to a tolerant time window For Threshold T1 , what presumably matters are the absolute amount of Emc and the rate of production of Ac and Sc, and the latter rate should depend on the amount of stimulatory “prepattern” factors [2891]. Because the variables are all constitutive (with no feedback until Step 4), the stoichiometry easily explains why the process is sensitive to gene dosage. What is not so easy to understand is why phenotypes remain wild-type when the AS-C dose is halved or doubled, or when the dose of emc is halved (Fig. 3.12). Why don’t such changes affect bristle number like greater alterations do (e.g., halving emc dose plus doubling sc dose)? Historically, the ability of animals to maintain phenotypic constancy in the face of perturbations (genetic or environmental) led to the related concepts of “buffering” [1748, 3709] , “canalization” [1470] , and “robustness” [2565, 3032, 4646] . Certainly, ﬂies do use a variety of error-correction strategies at every echelon (e.g., genes [4584] , proteins [2760] , cells [1292] , and tissues [12, 1940, 2527, 3042]), and many of these “quality control” tricks are undoubtedly sophisticated. However, the buffering of SOP number may

73

instead have a trivial explanation. To wit, there may be enough tolerance in the system to accommodate minor changes in AS-C activity by letting events shift along a time line (Fig. 3.12). If the slack is ﬁnite, then greater changes in AS-C activity will eventually push events to the edges of this time window, and no further slippage will be possible. At that point, the phenotype should begin to overtly reﬂect added deviations in genotype. Speciﬁcally, 1. Two-fold increases in AS-C dose (or halving the emc dose) might merely hasten Step 5 (before t2 ). Further ﬂooding of the system with Ac and Sc (i.e., changes greater than a factor of two) might accelerate the process so much that more cells can cross T2 and become SOPs before SOP inhibitory ﬁelds can extend to their maximal diameter – hence leading to greater bristle density. 2. Halving the AS-C dose should postpone the time at which Step 5 is reached. However, if upstream transactivators (Step 1) are still present, then the process might continue beyond t2 until it reaches completion. Further decreases might slow the process so much that the prepattern factors disappear before all the gaps between SOPs are ﬁlled in. This “Time Window Model” is buttressed by timing shifts of emc LOF SOPs [912, 1926, 2690]. It is also consistent with temporal changes in (1) N null SOPs [3689], (2) the CNS and PNS of N null embryos [379, 1563], and (3) SOPs in Egfr LOF mutants [917]. On the wing, SOPs of ectopic sensilla (caused by hLOF or ac GOF ) can arise within a broad time span and still develop normally [362], and the same is true for extra SOPs in PNCs of pyd LOF mutants [733]. Like late passengers missing a train that always leaves the station on schedule, these delayed pyd LOF SOPs make shorter bristles [3067] because cuticle secretion starts on time, and a similar constraint explains the fewer bristles that come from depleted histoblast nests [581]. Indeed, AS-C and E(spl)-C genes may be short and intronless so that their proteins can be made quickly and timed precisely [92]. Figure 3.13 summarizes the core circuit for bristle SOP selection. The idea that SOP inception is time sensitive may explain why it is temperature sensitive in hypomorphs [767, 769] not only during the PNC-SOP window [768], but also much earlier [770, 1997, 1998, 3405]. As Goldschmidt argued long ago [1520], shifts in the relative rates of reactions can uncouple key processes, leading to a later failure of the system to reach a critical threshold. The

74

IMAGINAL DISCS

a

Stages

(Prepattern) Ac Sc

and Hairy Emc

or

or 1. Competence

and

Da

Non-SOP

SOP

(Proneural) or

1 1 0

0

1

c

1 or

1

0

d

1 or

0

1

1 1

1

1 1

1

2. Fine-tuning

0

0

1

Non-SOP

Ac h

or

or

and 0

1 and 1

1 1

SOP

1 1

or

1 and 1 Non-SOP 1

and 1

or

1

1 or

1 1

1

1 and

0 1

1

1 or

0 0

1

and

0 0

0

1 1

0

1 0 0

SOP

1

and 0 0

SOP

1 and

or

or Dl Sca

b

SOP

Ac Sc

and Dl Sca

0

0 0

or

antecedent reactions for bristle patterning remain to be identiﬁed. A time window also constrains the number of SOPs that form the cluster of chordotonal receptors in the femur. In contrast to how bristle SOPs arise, chordotonal SOPs emerge from their PNC as a tandem chain via reiterative induction of new SOPs by old SOPs. The process starts and stops at deﬁnite times, and the number of SOPs can be increased or decreased by raising or

e

0

lowering the perceived volume of the inductive (EGFR pathway) or inhibitory (Notch pathway) signals during that window [4898].

Atonal and Amos are proneural agents for other types of sensilla Chordotonal PNCs differ from bristle PNCs insofar as they rely on the bHLH gene atonal instead of ac or sc [3181, 4897], and the same is true for photoreceptors in

CHAPTER THREE. BRISTLE PATTERNS

75

FIGURE 3.13. Circuitry that assigns SOP (bristle) vs. non-SOP (smooth cuticle) cell fates in the epidermis (cf. Fig. 2.7 for key to

symbols). a. Core pathway of proteins that decide SOP vs. non-SOP identities (see text for further details). Abbreviations: Ac (Achaete), Da (Daughterless), Dl (Delta), Emc (Extramacrochaetae), h (hairy), Sc (Scute), Sca (Scabrous). In the ﬁrst stage, “prepattern” factors (cf. Chs. 5 and 6) prompt the synthesis of Ac and Sc in certain regions by acting through cis-enhancers in the AS-C. When Ac and Sc (which act redundantly) heterodimerize with Da (which is ubiquitous), they endow a cell with the “competence” to become a SOP. This “proneural” state can be prevented by the antineural agents Hairy (which primarily affects Ac) and Emc, both of which act intracellularly (Fig. 3.12). In the second stage (shaded), one or more SOPs are selected by “ﬁne-tuning” within the proneural ﬁeld. Continued accumulation of Ac and Sc prods a cell to become a SOP, unless it is blocked by external antineural signals emitted by SOPs themselves. Dl and Sca are thought to be short- and long-range agents, respectively (see text), that create an “inhibitory ﬁeld” around each SOP. b–e. Schematics of cells (b–d) that are selecting various fates at different tissue locations (e). States of components (cf. a) are recorded as “1” (present) or “0” (absent), and black circles denote determining factors. b. The circuit as it functions in areas where Hairy is expressed (e.g., leg stripes, e). The cell never becomes competent because Hairy is the controlling factor. c. If a cell is impelled by prepattern factors (circled “1”) and no antineural agents are present, then it will complete the gauntlet and become a SOP. d. Any cell inside the inhibitory ﬁeld of a SOP cannot become a SOP. Sca (circled “1”) is here assumed to be the diffusible inhibitor. e. Rectangular piece of leg skin (Fig. 3.9c) containing one Ac stripe (shaded) and the adjoining Hairy interstripe (hatched). Among these ∼150 cells (hexagons), only 4 SOPs (black cells, c) will form. Remaining cells are prevented from becoming SOPs because they contain Hairy (b) or are inhibited by a nearby SOP that adopted this state (stochastically) before they could do so (d). Ovals mark limits of inhibitory ﬁelds, which are assumed to be anisotropic because the inhibitor (Sca?) does not diffuse freely. N.B.: In D. melanogaster, non-SOP cells look alike, regardless of whether they were previously proneural, but other species convert some proneural ﬁelds into pigment stripes [615]. Although the circuitry depicted here is digital, the “SOP computer” actually operates partly in an analog (threshold) mode (Fig. 3.12; see text). Also, it is not known whether Sca acts via the same pathway as Dl [113], and other questions remain about the core logic [178]. In principle, the “and” gate preceding Ac and Sc should cancel out the subsequent “or” gate, but this part of the circuit is included because Ac and Sc can be independently changed by mutations (Fig. 3.5) or various LOF-GOF manipulations (App. 5).

the eye disc [182, 1076, 2041, 2042]. Still another bHLH gene – amos – establishes PNCs for olfactory sensilla and leg claws [1587]. LOF and GOF studies have shown that these different bHLH genes not only set up PNCs, but also determine the identities of the sensilla that arise therein (cf. Ch. 2). For example, overexpressing Scute induces bristles only [912, 918, 2038], whereas overexpressing Atonal induces chordotonal organs [764, 2038, 2040], and overexpressing Amos elicits both chordotonal and olfactory sensilla [1587, 1928]. Thus, no clear distinction exists between proneural genes on the one hand and sensillumidentity genes on the other [448, 1928].

Other (upstream) pathways govern bristle patterning Polychaetoid is intriguing because it may link the HLH circuitry to the Notch pathway [4811] and adherens junctions [533]. The involvement of pyd in HLH affairs is indicated by (1) reduced transcription of emc in pyd LOF wing discs [4237], and (2) synergy of pyd LOF with emc LOF , hLOF , and ac GOF [733, 1802, 3068, 3069]. The dose-dependent synergy of pyd LOF with both NLOF and DlLOF is equally dramatic [733], and other clues also point to an afﬁliation

of pyd with the Notch pathway – viz., (1) Pyd’s location at junctions [4236] in the same apicolateral ring where N is concentrated (Pyd is homologous to mammalian Zonula Occludens-1) [1203], and (2) Pyd’s ability to bind the product of a gene (canoe) that also interacts with N (App. 5) [4236]. If pyd serves such a key role, then why don’t pyd LOF mutations affect all bristles equally? Why, for example, are extra MCs found in the dorsocentral area ∼10 times more often than in the notopleural area [733]? The spatial heterogeneity cannot simply be due to LOF allele quirks (a bugbear of the AS-C) [533], because it also occurs with null alleles [733]. This same question was raised for Hairless in Ch. 2, where it was argued that a second gene might share its function. Although a redundant agent might also solve the pyd problem, it is possible that pyd is part of a separate pathway that is upstream of the entire SOP siting program [2363]. Pyd may instigate certain PNCs (via “Pyd AS-C”), but genes in other pathways may establish the other ones. Such genes are the elusive prepattern factors that Stern tried so hard to ﬁnd. How they act is discussed in subsequent chapters.

CHAPTER FOUR

Origin and Growth of Discs

Insect imaginal discs are barely visible to the naked eye, so detailed observations had to await the invention of adequate magnifying lenses [1266, 3064]. Discs were ﬁrst described by the great naturalist Jan Swammerdam (1637– 1680) [821, 3422, 4586], a contemporary of Leeuwenhoek’s, who applied his training in human anatomy to the study of insect morphology [3133]. In his Book of Nature (printed in English in 1758), Swammerdam waxes lyrical about the metamorphosis (which he calls “mutation”) of appendages (“horns” are antennae) in hymenopteran larvae (“worms” of bees):

The wings, horns, and other parts which worms without legs seem to acquire about their chests at the time of their mutation are not truly produced during the period of mutation, or, to speak more agreeably to truth, during the time of the limbs shooting or budding out, but . . . have grown there by degrees under the skin, and as the worm itself has grown by a kind of accretion of parts, and will make their appearance in it upon breaking the skin on its head or its back, and thereby give it the figure of a nymph, which it would afterwards of itself assume. Hence it is, that we can with little trouble produce [by dissection] the legs, wings, horns, and other parts of an insect, which lie hid under its skin while in the shape of a naked worm, which has neither legs nor any other limbs. . . . The nymph . . . is nothing more than a little worm, which, the growth of legs, wings, and other limbs hid under its skin being perfected by time, at last bursts that skin, and casting it off, gives us a clear and distinct view of all those parts. This change . . . is no more mysterious or surprising than what happens when one of the meanest plants, despised and trodden under foot, gradually swells on every side, and after producing a bud, by bursting the little case containing it, presents an elegant and beautiful flower. [4218]

76

The ﬁrst account of discs in dipterans appeared in 1864 when August Weismann (renowned for his germ plasm theory [1487]) analyzed the stages of development in Musca and Sarcophaga [4586]. In 1936, after D. melanogaster had become the darling of genetics, a former student of Spemann’s, Charlotte Auerbach [2215, 4696], traced the development of leg, wing, and haltere discs back to the newly hatched larva [142]. Dietrich Bodenstein studied the development of all the discs for Demerec’s Biology of Drosophila (1950) [1019], which served as the standard reference for a generation of ﬂy researchers. The precursors of discs within the embryonic blastoderm were mapped in the 1970s by microcautery [425], microbeam irradiation [2591], and cell-lineage techniques [1695, 2026, 4652]. Each disc was found to come from a distinct cluster of blastoderm cells (Fig. 4.1).

Segmentation genes set the stage for disc initiation Not until the genetic basis of segmentation began to be analyzed ca 1980, however, was it possible to discern the factors that cause discs to originate. In that year, ¨ Christiane Nusslein-Volhard and Eric Wieschaus deﬁned the hierarchy of “segmentation genes” that divides the embryo’s anterior-posterior (A-P) axis into metameres [2178, 3151, 4069]. The hierarchy has four echelons (Fig. 4.2) [2717, 3248, 3843, 4671]: 1. Axis genes (a.k.a. “maternal-effect” genes). Three proteins form gradients along the A-P axis: “Bicoid” is made from mRNA maternally deposited at the anterior pole [1104] , “Nanos” comes from posterior

CHAPTER FOUR. ORIGIN AND GROWTH OF DISCS

A7

A1

T3

T2

77

T1

3

H

1

8

4

2

10

9

Discs

as T1 T2 T3 2 T 3 A1 1T T dEpi dEpl

blastoderm fate map

A7

H1

pc pmg

H2

vNR amg

H3 H4

D A

H5 H 6

mes

P

1 2 3 4 5 6 7 8 9 10

Labial Clypeolabral Humeral Eye-antennal 1st Leg 2nd Leg 3rd Leg Wing Haltere Genital

14 cells 100 µm

V

5

6

7

FIGURE 4.1. Origin of discs as “islands” within the embryonic ectoderm. Left side of an embryo (below) at the cellular blastoderm stage (D, dorsal; V, ventral; A, anterior; P, posterior). Spots indicate where discs (key at right) or histoblast nests later form. In this fate map, segments (straight or warped rectangles) are numbered in each region (Head, Thorax, Abdomen). Areas shaded medium (dEpi, dorsal epidermis) or dark (vNR, ventral neurogenic region) form larval skin, except that ∼25% of vNR cells ingress as neuroblasts. Gastrulation internalizes the midgut (amg, pmg), mesoderm (mes), pole cells (pc), and virtually the entire head [4421]. Amnioserosa (as) is extraembryonic. This fate map is based on Hartenstein’s atlas [1739], with the following exceptions: (1) D midline is not tilted toward viewer; (2) clypeolabral, eye, labial, and humeral sites are as per gynandromorph maps [1695, 1777, 2026, 4146], although the actual humeral site may be more ventral because its spiracle and the 1st-leg disc can share blastoderm cell ancestors [237, 928, 2819, 4565]; (3) eye anlage is shown as one oval instead of the three spots that Volker Hartenstein used (pers. comm.) as an arbitrary way to connote the eye disc’s mysteriously diffuse origin [1147, 1224, 2103] and its patchy apoptosis [3058, 4825]; (4) thoracic discs are more dorsal as per cell transplant data [2820]; (5) wing disc and 2nd-leg disc are fused as per histologic [827, 834] and lineage data [2819, 4076], as are haltere disc and 3rd-leg disc; and (6) genital disc spans A8–A10 [429, 735, 2343]. N.B.: H1–H3 are stylized [1739] and probably not contiguous [832, 1300, 1631, 2104, 4825]. See [2103, 3058, 3631] for head and tail details and [631, 3791, 3792] for other nuances. In the schematic drawing of the larva (above), discs are spread out and the midsection is omitted. See also App. 7.

mRNA [939] , and “Caudal” is made from uniformly distributed mRNA whose translation is repressed by Bicoid [3116] . Among these three factors, only Bicoid appears to be a bona ﬁde morphogen (see below) [1945, 2886, 4154, 4163]. The division of labor between maternal and zygotic (gap, pair-rule, and segment polarity) genes [2105, 3152, 4653] makes sense evolutionarily, given the time constraints of embryogenesis [4650]. 2. Gap genes. During the syncytial blastoderm stage, genes in the “gap” class are expressed in broad (∼10– 50% egg length) bands [1398]. They are so-named because LOF mutants are missing large parts of the segment series. Some initial overlaps between ex-

pression bands are erased by mutual repression [2329, 3567]. 3. Pair-rule genes. Spatial periodicity ﬁrst appears when genes of the “pair-rule” class are transcribed. Their name comes from an absence of alternating segments in their LOF mutants. This echelon, which came as a surprise [659, 3248], is divisible into primary and secondary tiers [1750, 3248]. Primary pair-rule genes integrate nonperiodic cues from axis and gap genes (with some inter se inputs), and most of their expression stripes (∼7 per gene) are separately regulated by a combination of upstream factors [242, 1722, 1750]. Secondary pair-rule genes rely more on the periodic outputs of primary genes. For example, fushi tarazu

78

IMAGINAL DISCS

Percent Egg Length

gap

90

80

70

60

50

40

30

20

10

bcd Bcd

0 Bcd

Cad

cad

Hb

kni

Kni

gt

pair rule

Kr eve ftz

Ftz

en wg Antp

homeotic

segment identity

100

hb

seg. pol.

segmentation gene hierarchy

axis

a

D

Ubx

A

P V

abd-A Abd-B

b Parasegments

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Segments H4 H5 H6 T1 T2 T3 A1 A2 A3 A4 A5 A6 A7 A8 A9 FIGURE 4.2. Categories of genes involved in body segmentation and segment individuation.

a. Protein levels (Bcd, Cad, Hb) or mRNA levels (remainder) detected or inferred [3605] for a few members of each class in the segmentation gene hierarchy (axis, gap, pair rule, segment polarity), or (below) expression patterns of key homeotic genes at the time when their activity is instrumental in patterning [3248]. Gene abbreviations: abd-A (abdominal-A), Abd-B (Abdominal-B), Antp (Antennapedia), bcd (bicoid), cad (caudal), en (engrailed), eve (even skipped), ftz (fushi tarazu), gt (giant), hb (hunchback), kni (knirps), Kr (Kruppel), Ubx (Ultrabithorax), wg (wingless). Embryos at right depict realistic Bcd, Kni, ¨ and Ftz protein distributions [105, 661, 1102, 1103, 4055]. The Cad gradient arises via Bcd repression of cad mRNA translation [1487, 3116]. Vertical lines show a few other interactions: (1) boundaries of eve stripe 2 (arrowheads) are deﬁned by gt and Kr [4764]; (2) even-numbered en stripes (arrowheads) emerge due to a trailing gradient of Eve that turns off odd skipped (not shown) in the front cells of each ftz stripe (thus activating en by repressing its repressor) [877, 1324, 2681, 2682]. See [1605] for a comparable chain of negative regulators and [2002] for Deformed, where the control is more combinatorial than hierarchical. b. Embryos make 14 parasegments and 15 segments, with an en stripe (shaded) at the front or rear of each, respectively. Panel a is adapted from [162, 1416, 1946, 3605], with eve stripe 2 and en stripe 4 data from [4764] and [1324]; b follows [3843] (“A9” stands for A9–A10). N.B.: As a “wiring diagram,” this illustration is woefully inadequate because it omits so many key genes [47, 380, 1962, 3248], spatial details (e.g., uneven levels within homeotic gene domains [682, 2717]), and dynamic modulations [242, 1790, 2181, 3770, 4834]. Nevertheless, at least it conveys a feeling for the ﬂow of control. See [1487, 2121] for excellent exegeses and [2869, 4893] for evolutionary context.

CHAPTER FOUR. ORIGIN AND GROWTH OF DISCS

has a compact “zebra” cis-enhancer that governs all 7 stripes [4834] . 4. Segment polarity genes. At the cellular blastoderm stage, the foregoing factors turn on the “segment polarity” genes. Their name comes from the segmental periodicity of LOF defects and associated reversals in cuticular polarity. Among them, engrailed and wingless are instrumental in setting metamere boundaries. Bicoid and Caudal are transcription factors [1011, 1709], as are all gap and pair-rule gene products [3768] , whereas Nanos acts on mRNA processing [4756]. These roles make sense because early mitoses are syncytial, which allows for free diffusion of signals among nuclei [1962]. By the time pair-rule stripes sharpen, however, plasma membranes are partitioning the cortical nuclei into a cellular blastoderm [47, 657, 3843], and direct internuclear communication is no longer possible [1429]. Not surprisingly, segment-polarity genes encode all sorts of proteins involved in transducing signals from the cell surface to the nucleus [3192, 3341]. Only two signals are critical here: Wingless and Hedgehog [1010, 1167, 4499] . Ultimately, the segmentation gene hierarchy regulates expression of “Hox” genes, which implement segmental identities (cf. Ch. 8 and App. 1) [2002] . Control of Hox genes stems mainly from the gap gene level, but inputs come from other levels as well (cf. Fig. 8.1) [683] . The overall circuitry is complex because many of the ∼50 core segmentation genes interact with ≥5 other such genes in the same or different echelons [1487, 2121] . Moreover, in some cases, the links skip a level or go back up the chain of command [3815] . Details are still being worked out [3383] , but the main design features are now clear [968] : 1. Both analog and digital controls are employed, and the driving inputs can be positive or negative [657, 2446] . 2. Binary (on or off) states are stabilized by positive feedback [2057] and arise, at the DNA level, via cooperative binding of trans-acting factors at cisenhancers of target genes [1708] . 3. Inputs are typically processed by combinatorial logic (as “and” gates) at these cis-enhancers [414] , while other aspects of Boolean syntax are mediated by competitive binding [3993] , dimerization [3768] , quenching [1600] , physical spacers [3992] , and “dual control” [657] .

79

These elements mesh to form a dynamic and robust system [4499] that orchestrates thousands of target genes [2541] . Despite its virtuosity, however, this “gene machine” is not optimized [4670] since certain pairs of components (nanos and hunchback [1945, 1986, 4154] or odd skipped and engrailed [878]) can be removed with little or no effect on the patterning of denticle belts. Aspects of the system have been modeled mathematically [2811, 3704], but computer models are best for tracking its behavior in real time. Simulations by John Reinitz [3567] and others [380, 2200, 2201] are yielding more testable predictions as additional parameters of the components become known [1010]. By 1987, the segmentation gene network was coming into focus, and seminal reviews were authored by Philip Ingham and Michael Akam [47, 1962]. Tellingly, these normally reserved British dons could barely contain their excitement at humankind’s ﬁrst glimpse into the molecular clockwork of pattern formation. Indeed, the insights galvanized the whole ﬁeld [125, 3669]. Akam realized that the embryo’s strategy of making segments from overlapping stripes fulﬁlled Curt Stern’s vision of prepatterns [47]: The pattern generated by the gap and pair-rule genes is transient and can appropriately be described as a prepattern.

As discussed in Ch. 3, Stern thought that prepatterns are transient networks whose interdependent nodes evoke elements in the ﬁnal pattern [4100, 4346]. In this case, the nodes would be the boundaries that delimit metameric regions along the A-P axis [657, 2433], and their interdependence would be attributable to the circuitry of the segmentation gene hierarchy [3248]. Ironically, Stern’s Prepattern Hypothesis had been toppled as the ﬁeld’s reigning paradigm in 1969 by Lewis Wolpert’s “Positional Information Hypothesis” [4724]. Wolpert argued that patterns are organized by global coordinate systems instead of by skeletal frameworks (see below). Akam’s remark heralded the resurrection of the prepattern concept as a useful way of thinking about embryos and discs [2446, 3953], although its full rehabilitation took several more years. To contrast these distinct schools of thought, the history of their conﬂict is brieﬂy recounted below. Although retrospective, the next section is not antiquarian, and readers would do well to study it. It constitutes the bedrock for the rest of the book.

80

Prepatterns and gradients clashed in trying to explain homeosis Stern’s reasoning was straightforward. If prepattern genes establish the prepattern’s nodes and the nodes are interdependent, then disabling one or more nodes within a LOF mutant clone should cause the remaining nodes outside the clone (in wild-type terrain) to redistribute themselves. In other words, any prepattern gene would have to behave nonautonomously in genetic mosaics [4100]. Today we know many such genes, most of which encode diffusible signals, but at that time (1954) none were known. Stern and his associates tested one mutation after another to see whether they could alter patterns nonautonomously in mosaics. By 1967, they had assayed ∼20 genes but had not yet found a bona ﬁde case of pattern reorganization [4346]. In 1967, Stern’s long quest for a “prepattern mutant” was rewarded when he and Chiyoko Tokunaga tested eyeless-Dominant (ey D ) in mosaics and found that it acted nonautonomously [4100, 4346]. Although its name comes from its eye defects (a result of cell death [1309]), this mutation also causes extra rows (∼2–6 rows vs. one in the wild-type) of sex comb bristles (∼39 vs. ∼10 bristles in the wild-type) on male forelegs. In mosaics, wild-type cells were found to be able to participate in extra rows when they intrude into the sex comb area of an otherwise mutant leg [4109]. Apparently, ey D augments the sex comb node or “singularity” in the leg prepattern, and wild-type cells respond accordingly – i.e., “ey D mutation bigger sex comb singularity phenotype” (“ ” denotes “causes”). However, other aspects of the mutant syndrome are visible in forelegs of both sexes and hence cannot be speciﬁc for the sex comb: swelling of the distal basitarsus, extra bristles of other types, disruption of pattern and polarity, and fusions of 1st and 2nd tarsal segments. These additional defects suggested other etiologies [1421, 1799]: overgrowth 1. Overgrowth Scenario: ey D mutation more sex comb positional coordinates phenotype [3213, 3448, 4348, 4724]. Conceivably, ey D induces overgrowth by disrupting Sex combs reduced [3332, 3333] – a Hox regulator at this spot. 2. Segmentation Scenario: ey D mutation gap in intersegmental membrane distorted gradient more sex comb positional coordinates phenotype [523, 2427, 3427, 4100, 4517]. This argument is predicated on the assumption that the ﬂy leg uses a sawtooth series of segmental gradients to specify positions along its

IMAGINAL DISCS

proximal-distal axis [385, 386, 1801, 2425]. Interrupting the barrier between the low end of one gradient and the high end of the next one (i.e., intersegmental membrane) could cause a “backﬂow” of morphogen, with the consequences listed above [2423]. 3. Apoptosis Scenario: ey D mutation cell death compensatory overgrowth phenotype [1314, 3441, 3442, 3964, 4346] (see [4336] for a striking phenocopy). Despite the fact that we still do not know which of the above explanations (if any) is right, ey D gave a ﬁtting ending to the prepattern saga, and Stern featured this story in his 1968 opus Genetic Mosaics and Other Essays [4100]. The celebration was short lived, however, because Wolpert proposed his model in the next year. The coup de grace that killed Stern’s model was not the paucity of prepattern mutants, which he rightly attributed to lethal side effects that would preclude their survival [4100]. Rather, it was the fact that homeotic mutations act autonomously [526, 1357, 4346]. “Homeotic” mutations, by deﬁnition, transform particular body parts to resemble other body parts – e.g., a leg into a wing (cf. Ch. 8) [2509, 3214, 4486]. Assuming that each organ has its own prepattern, Stern expected mosaic organs to display signs of jousting prepatterns – nonautonomous inﬂuences of homeotic tissue on nearby wild-type cells or vice versa [4097, 4098]. By 1968, however, several contradictory cases had been documented. For example, extra sex combs LOF partly converts 2nd and 3rd legs into 1st legs [1713], yet acts autonomously in mosaics [4349]. This result was not too unsettling for the hypothesis because all leg cells might “speak” a common language. No such rationalizing was possible, however, for bithorax LOF (anterior haltere into anterior wing) [2506] or aristapedia LOF (distal antenna into distal leg) [3444, 3614] because it seemed inconceivable that the same scaffolding could be used for organs as different as halteres and wings or legs and antennae (Fig. 4.3) [3214]. In his 1968 book, Stern acknowledged this paradox but did not offer a solution [4100]. In a review of Stern’s book, Peter Bryant argued that stretching the prepattern concept to cover these situations undermined its usefulness to the point of absurdity [520]. Some workers, including Stern, have employed the concept [of prepattern] as specifying, in Drosophila, individual bristle positions. However, experiments using mosaics for homeotic mutants tend to indicate that the prepattern for antennal structures is identical to that for leg structures. In that case, the prepattern could not be specifying individual bristle

CHAPTER FOUR. ORIGIN AND GROWTH OF DISCS

positions since the bristle patterns are entirely different in these two appendages. If the concept must be generalized to such an extent that the prepattern for leg is identical to that for antenna, then it seems to lose much of its usefulness as a working hypothesis. We are left, in fact, with the rather hackneyed interpretation of pattern formation as resulting from a gradient of some hypothetical morphogenetic influence. [520]

A new conceptual framework emerged in 1969 when Lewis Wolpert blended Hans Driesch’s old notion of coordinate systems [1101, 3741, 3742] with modern information theory [95, 2547, 2766, 2989] to create the idea of positional information (PI) [4724, 4731, 4734]. It was not this homeosis problem that PI aimed to solve, but rather the “Regulation Riddle”: how can patterns robustly regenerate? Nevertheless, an explanation for homeosis followed naturally. Indeed, Wolpert cited the autonomy of aristapedia LOF as support for the “universality” corollary of his theory – namely, that the same PI coordinates should be readable by cells of any histotype. Coincidentally, in that same year Wolpert was handed an even better example. Antenna-to-leg homeosis in Antennapedia GOF (Antp GOF ) affects not just the arista but the entire antenna, and the patchiness of the transformation in nonmosaic mutant ﬂies revealed a striking correspondence between antennal and leg regions (Fig. 4.3a) [3445, 4728]. Such different organs could not possibly share a common prepattern (or so it was thought), but they might easily use a common coordinate system. The Prepattern Hypothesis seemed doomed. For me, the most significant contributions to the study of pattern formation over the last 30 years come from the work of Stern on genetic mosaics and the concept of prepattern. . . . This work provides excellent evidence for the concept of positional information and polarity potential, and the best evidence for the postulate of universality, at least within the same animal. As will be seen, the concept of positional information gets over some of the difficulties associated with the concept of prepattern. . . . Along similar lines one can interpret homeotic mutants which involve genes such as aristapedia which cause antennae to form legs. Once again the positional information may be the same and only the interpretation different. This is in line with the postulate of universality and it is thus again encouraging to find genetic mosaics of aristapedia with normal tissue behaving [autonomously] according to position and genome. [4724]

Wolpert invoked three stages for patterning and used a French Flag to abstractly represent three distinct cell types [4723]:

81

1. Speciﬁcation of PI. A scalar variable changes linearly or exponentially along an axis [3989] (e.g., the activin gradient [1656, 4220]). In the archetypal model, the variable is a “morphogen” (diffusible signaling molecule) whose concentration describes a “gradient” across the “ﬁeld” of cells whose fates it “speciﬁes” [4732]. Ambiguities in these terms can be minimized by operational deﬁnitions [967, 4673] (e.g., a “morphogen” is a signal that elicits expression of different genes at different concentrations [865, 1655]). Morphogens were imagined to emanate from a terminal source, with or without a sink where the morphogen is degraded [210]. Gradients are usually assumed to reach equilibrium before being “read” [3074], but this need not be true [2780, 3225]. Also unclear is whether cells “ratchet” to a particular level of response whence they are unable to retreat [1657]. In theory, cells can tell the absolute size of a ﬁeld and their orientation therein by sensing slopes of gradients [2434, 2448]. Finally, there is the issue of resolution (signal-to-noise ratio) as a function of distance. For example, in ﬂy eyes, the signal for ommatidial polarization is sensed over distances of >100 cells with 100% ﬁdelity (cf. Fig. 7.5) [2883], but how? 2. Recording of PI. To gauge its position, each cell measures the local height of the PI variable – like a person guessing the nearness of an ambulance by the loudness of its siren. (How ﬁnely cells can measure concentration differences is unknown [1142, 1655, 4366].) Cells then record this information (e.g., as on or off states of “memory” genes) [1608, 1964, 2417, 2515, 2803, 4730] as a function, perhaps, of receptor occupancy on the cell surface [1129, 1655, 2779] or of sensitivity thresholds in target gene promoters [1284, 1923, 2678, 3087, 3406]. The gradient itself can then disappear since it is no longer needed. How long such memories can persist in dividing cells is unclear. 3. Interpretation of PI. At a later time, cells translate the memories of their old locations (“positional values” [4729, 4730, 4732, 4734]) into differentiated states (blue, white, or red) through some sort of downstream gene circuitry [98, 2646] – like a letter carrier deducing city names from postal codes (e.g., see [472]). Whereas Step 2 converted analog to digital information, Step 3 must convert quantitative to qualitative states [1655], although memories may also control analog traits such as adhesivity [2436] or mitotic rate [1263]. One abiding mystery is the extent to which neighboring cells interact to sharpen the interpretation zones [1143, 1609, 4691].

IMAGINAL DISCS

antenna

xi m

ro

p

A2 A3 Co

T1

b Models

Fe

leg

Positional Information Hypothesis 7 6

Gra

dien

4 3 2 1

1. Specification

t

? ? ? ? ? ? ? subregions 2. Recording

7 6 5 4 3 2 1 3. Interpretation

(A-type)

positional values

(L-type)

pattern

Ar BC

A2 A1

A3

antenna

x T5 T2 T1 Ti Fe Tr Co Cl T3 T4

leg

Prepattern Hypothesis 1. Prepattern 2. Competence

singularities

x (A-type)

(L-type)

pattern

x

antenna

leg

xi m

D

Ti

[M] 5

V distal

T4 T3 T2

Tr

ro

BC T5

V

al

Cl

A1

Ar

distal

a Homology map

D

al

82

p

CHAPTER FOUR. ORIGIN AND GROWTH OF DISCS

PI and prepatterns are often contrasted in terms of how they might create an array of evenly spaced bristles within a line of cells [526, 2764, 4671]. PI would use a gradient to assign a number to each cell, and certain numbers would signify “bristle” because the genome is “wired” accordingly. A prepattern strategy, on the other hand, would use some sort of device (e.g., reactiondiffusion) to put bristle-inducing signals at regular intervals, and any cell would be able to respond (above a certain threshold). With PI, every cell knows where it is and differs from every other cell, whereas prepatterned cells would know their state (bristle vs. nonbristle) but not their location [4725, 4726, 4729].

83

In the PI scheme, patterns are essentially encoded as bitmaps [1805, 3610], with each cell (≈pixel) having a separate genetic representation (≈on/off state). The huge coding capacity needed for intricate patterns (even with combinatorial encoding) strained credulity [862, 2142, 2646], as did the paucity of plausible molecular mechanisms [1805, 2809, 3087, 4730, 4732], but neither quibble slowed the PI model’s rise to popularity [864, 3988, 4734]. It quickly attained paradigm status. Stern’s camp formally “surrendered” in 1978 [4346]. Soon thereafter, direct support for PI came from the segmentation genes [4733]. They furnished the ﬁrst deﬁnitive morphogen (Bicoid) [1103] , plus insights into

FIGURE 4.3. Demise of the Prepattern Hypothesis in 1969 and ascendancy of the Positional Information Hypothesis, due in part to their clumsy or elegant explanations for homeotic autonomy. a. Correspondence (arrows) between antennal and leg parts, as deduced from Antennapedia R (Antp R ) ﬂies, whose antennae are patchily transformed into 2nd legs [3445]. Abbreviations: A1–3 (antennal segments 1–3), BC (basal cylinder), Ar (arista), Co (coxa), Tr (trochanter), Fe (femur), Ti (tibia), T1–5 (tarsal segments 1–5), Cl (claws). Compasses denote adult polarities (D = dorsal, V = ventral; the inversion is explained below). Antennae of Antp R ﬂies (not shown) have patches of leg tissue. Whenever leg tissue arises at a particular proximal-distal level, it makes leg structures appropriate for that level. The implication is that these leg cells can somehow ascertain their location along this axis of the antenna. Wolpert and Stern agreed that the antenna and leg probably share some kind of “ground plan” that both types of cells can “read.” They disagreed over the kind of ground plan. In Wolpert’s scheme, it is a gradient; in Stern’s, it is a prepattern. b. The two models. Each of the 7 gray rectangles represents a segmental region (eventually containing 102 –103 cells) of the antennal or leg rudiment along the proximal-distal axis. Certain segments are subdivided or grouped. Lewis Wolpert’s Positional Information (PI) Hypothesis explained antenna-to-leg homeosis in terms of a shared coordinate system [4724]. Antennal and leg cells are supposed to use the same positional signal – a diffusible molecule “M.” This “morphogen” is produced at one end of the organ (left) and diffuses to form a concentration gradient (triangle). The gradient could be linear (as shown) but is more likely exponential. In Step 1, cells assess their distance from the source by measuring the amount of M: the more M that a cell “tastes” at its site, the nearer it “thinks” that it must be to the source. In Step 2, cells record these levels (1–7) as “positional values” (on or off states of memory genes?) that persist after M vanishes. In Step 3, cells translate these values into structures that suit the disc to which they belong. For example, antennal cells interpret the number 7 as “arista,” whereas leg cells interpret it as “T5 and claws.” The interpretation mode that a cell uses (A vs. L) is predetermined by the turning on or off of a particular homeotic gene in one of the cell’s ancestors. Mutations in that gene cause homeosis: mutant cells “think” they are leg cells, but they can still sense M properly, so they make leg structures that are appropriate for their proximal-distal level in the antenna. Finer-grain patterning within subregions may rely on a second echelon of (segmental) gradients (not shown) [386]. The elegance of Wolpert’s model has been its universality: the same gradients can theoretically pattern all the discs [20, 523, 3701]. Curt Stern’s Prepattern Hypothesis had a more difﬁcult time explaining homeosis. Its basic assumption was that every pattern element is preceded by a discrete signal (“singularity”) [4095]. Unlike the morphogen, these signals (circles, ovals, square, triangles, and “x”) differ qualitatively: they are not graded. In Step 1, the disc lays out an array of these signals (the “prepattern”). The autonomy of the Antp R mosaicism implied that antenna and leg must express one another’s signals. (How else could nouveau-leg cells that ﬁnd themselves “stranded” in the antenna ﬁgure out what structures to make?) In other words, both prepatterns must coexist in each disc [4346]. Antennal cells would normally read one subset of signals (white symbols), while leg cells read the other (black symbols). The ﬁltering of these subsets (cf. Fig. 3.2) would depend on a cell’s “competence,” which in turn would be determined early in development when one of the cell’s ancestors turned a particular homeotic gene on or off. Mutations in such genes would lead to interdisc transformations (cf. Ch. 8). This argument was tolerable for a single type of transformation (e.g., antenna-to-leg), but the large number of interdisc homeoses [3214] implied that every disc must contain the prepattern for virtually every other disc. Although not impossible, this notion seemed implausibly clumsy, especially when contrasted with how easily the PI Hypothesis handled this same phenomenon. Panel a is redrawn from [3447], which depicts a right antenna and left leg (see [1587] for A3 subregions, [1516, 1561, 2287] for molecular “homology,” and [4522] for a different map). Panels b and c are based on the ideas of Stern (Fig. 58 in [4100]) and Wolpert [4734], as contrasted inter se by Bryant [526] and Tokunaga [4346]. Black or white symbols in the antennal and leg schematics are merely abstract representations of the actual pattern elements. See Figure 8.3 for data on how antennal vs. leg identities are actually controlled and [2956, 3744] for the origin of the gradient concept. See also App. 7.

84

(1) how gradients can specify zones (e.g., gap gene enhancers varying in Bicoid-binding afﬁnity) [1105] , (2) how boundaries can be sharpened (e.g., competitive and cooperative trans interactions) [575] , and (3) how interpretation can be implemented (e.g., morphogen sensitivity being dictated by distance from cis-enhancer to core promoter) [1835, 2421]. The first step involves the localization of a cytoplasmic determinant, in this case the messenger RNA of . . . bicoid in the unfertilized egg. On fertilization the localized RNA becomes translated into bicoid protein, which spreads by diffusion and forms a morphogenetic gradient. The bicoid protein concentration provides positional information to the nuclei in which it is taken up. The third step involves the conversion of the graded pattern into a repetitive pattern of stripes under the control of the segmentation genes. Finally, the repetitive pattern is converted into a sequential pattern; the segments differentiate and each acquires its own identity [via] the homeotic genes. [1416]

Gradients and threshold responses appear to be used not only in Bicoid’s control of gap genes [1284] , but also in cross-talk among gap genes [4764] , in gap gene control of pair-rule genes [1323] , in cross-talk among pair-rule genes [4553] , and in pair-rule gene control of segment-polarity genes [1324] . Despite the network’s reliance on gradients, the way that it processes information is more reminiscent of prepatterns [2448]. To wit, cells only use certain Bicoid levels to establish zones of gap gene expression, and the zone borders act like singularities to delimit pair-rule stripes. The remaining levels are ignored. In a pure PI scheme, all levels should be recorded as heritable positional values [3087], but Stage 2 of the PI scenario seems to be bypassed. Thus, the process seems more like a PI-prepattern hybrid than strictly one or the other. Peter Lawrence, like Michael Akam, realized that these theories could dovetail nicely into a uniﬁed explanation [2446]: [The prime function of pair rule genes is] to locate boundaries that delimit fields or gradients of positional information. [2433]

Within each segment, cell positions were thought to be speciﬁed by a “segmental gradient” [2426] , based on a long history of surgical experiments [1300, 2430] . The segment-polarity echelon fulﬁlled this prophecy insofar as its stripes have a segmental periodicity [2717] , although the initial metamerism consists of “parasegments” [1978, 2438, 2724] that are out of phase with segment boundaries (Fig. 4.2b) [1772, 4152]. Two protein products of segment polarity genes – Hedgehog and Wingless – act like PI morphogens in

IMAGINAL DISCS

embryonic body segments [1789, 2448, 2716, 3748] and adult abdominal segments [2301, 2303, 3905, 4157, 4158] (see [2649] for a prescient model). Early experiments seemed to refute Wingless as a morphogen in the embryo [3730], but this conclusion was overturned by later studies which showed that sloppy paired imposes an uneven “landscape” of cellular competence to respond to Wg [596, 3129].

Homeotic genes implement regional identities How could identical gradients (segmental or parasegmental) produce different structures? According to PI theory, they must use different “code books” for interpreting their coordinates. In terms of Wolpert’s ﬂag metaphor, each segment would use the same coordinates, but Segment T1 might employ a British Flag interpretation mode, while Segment T2 uses an Italian Flag mode, etc. Only a few bits of information would be required to establish 15 or so interpretation modes along the A-P axis (one per segment). The process whereby segments become different from one another (“individuation”) is genetically separable from segmentation, because certain homeotic mutants undergo normal segmentation with virtually no individuation [2507, 4147]. What would happen if the interpretation mode of a particular segment were to malfunction? The segment should develop like another segment (i.e., undergo homeosis), and the same is true for interdisc transformations [1418]. Thus, the phenomenon of homeosis ﬁt neatly into the PI paradigm [523, 3701]. Presumably, homeotic genes would serve three PI-related functions: (1) use their on or off states to record various levels of the whole-egg gradient, (2) perpetuate these memories during disc growth, and (3) set the interpretation mode for each segment or parasegment [1365, 2755, 3882]. These expectations turned out to be basically correct (cf. Ch. 8). As Wolpert’s ideas were rippling through the research community in the early 1970s, a complementary model of Stuart Kauffman’s was also gaining notoriety [2155, 2156]. Its formulation for disc identities was based on fate switches observed during “transdetermination” – a sporadic metaplasia seen during long-term culture of disc tissue in vivo [1668--1670] or in vitro [3883, 3885]. Kauffman was struck by the fact that each disc only transforms into a few other types (cf. Fig. 6.9d). He argued that (1) these limitations are indicative of an identity code, (2) the code is binary, and (3) the “transition rules” (i.e., which disc can transform into which other discs) apply equally well to homeosis [2153, 2164, 3214]. According to

CHAPTER FOUR. ORIGIN AND GROWTH OF DISCS

his “Binary Code Conjecture,” each disc follows a preferred sequence of transdeterminations because only one bit changes at a time (i.e., 0 to 1, or 1 to 0). For example, if the code for 2nd leg is “1110,” then single switches could produce 0110, 1010, 1100, or 1111, but not other states (e.g., 1001) [2158, 2159]. Transdetermination and homeosis share other features [2082, 2159, 2754, 3788, 4142], although differences do exist [2138, 3448, 3883], and there are other possible explanations aside from coding (cf. Ch. 8) [1472, 2411]. In 1975, Antonio Garc´ıa-Bellido proposed his “Selector Gene Hypothesis” [1358]. Like Kauffman’s conjecture, it invoked switch genes for regional identities [701, 1635, 2930, 3457], but the regions were lineage compartments within discs rather than entire discs [2431]. At the time, it seemed conceivable that a succession of compartments might subdivide discs down to the single-cell level [1358, 2142, 2440], so that each cell would acquire an “area code” tantamount to a Wolpertian coordinate [1369, 1477, 3914]. However, that idea proved false [354, 494, 1375, 1639, 4671]. The discovery of the homeobox, announced by several labs in 1984 [2783, 2785, 3844], provided dramatic support for the selector gene idea [1412--1414, 2421]. This ∼180 b.p. sequence encodes a ∼60 a.a. DNA-binding domain (cf. App. 1) [1417, 1420]. As homeoboxes kept showing up in one cloned homeotic gene after another, it became clear that these genes constitute a distinct class structurally and functionally [1418, 3556] as contrasted with “housekeeping” genes whose products (e.g., metabolic enzymes) are used ubiquitously [293, 1865]. Since then, more evidence has surfaced for “master genes,” but their roles defy all the early models. Because it is not possible to understand how homeotic genes act without delving into each disc’s idiosyncrasies, further discussion is deferred until Ch. 8. One quirk of homeosis itself, though, merits mention here – the “Collective Amnesia Conundrum” [1805] (cf. “homeogenetic induction” [1005] and the “community effect” [4063]). Most models presume that disc states are carried by individual cells. A leg disc cell, for example, would know that it belongs to a leg disc and not to some other disc, regardless of its environment. This axiom was based on autonomy in genetic mosaics (see above) [2922, 4346] and retention of histotypes by dissociated disc cells in mixed aggregates [1374, 1411, 1421, 3138]. However, the islands of leg tissue in Antp GOF antennae arise nonclonally [3445], as if neighboring cells jointly forget their disc of origin, and transdetermination involves the same sort of process [1353, 1405, 1406]. Social cell behavior like this might depend on the nonclonal clus-

85

ters of synchronously dividing cells that are often seen in normal discs [23, 532, 2744, 2848] if (1) disc cells normally forget their identity at some stage of the cell cycle [4882], (2) they recover a sense of who they are by “reading” the identities of neighbors when they exit mitosis [533], and (3) too many cells simultaneously undergo mitosis, in which case groups of cells would remain amnesic and be forced to adopt a default state. Excess growth is indeed correlated with transdetermination [1421, 1670, 3883, 4334] and with “homeotic regeneration” [1421, 3448, 4142], but this “Cyclic Amnesia Scenario” is too simple [3448] because it predicts 1. Only one default state should exist per disc; however, each disc can actually transform into several other types [1421, 3214, 3881]. 2. Tumorous discs should exhibit frequent fate switches in situ; however, they typically do not [541, 1386, 2107, 3887]. 3. Fate switches should occur in normal discs by chance alone. On the contrary, homeotic outgrowths are virtually never seen in wild-type ﬂies [3883]. Observed cases of communal conversion may arise from diffusion [4063, 4563]: if single cells mistakenly start expressing a morphogen, then it could diffuse and activate inappropriate gene circuits so that unrelated neighbors jointly switch their fate [2082, 2754] (cf. Ch. 8). The dependence of transdetermination on wounding is consistent with this notion [3887] because wounding can evoke morphogens [489], and some homeoses are associated with tissue loss [839].

Wing and haltere discs ‘‘grow out’’ from 2ndand 3rd-leg discs Rudiments of the thoracic discs become morphologically recognizable when they invaginate after germband shortening (St. 13) at 9–10 h AEL (After Egg Laying, 25◦ C) [237, 2717, 2820]. At this time, the wing and haltere discs contain ∼24 and ∼12 cells, respectively [237, 664, 827, 2335], excluding mesoderm cells [57, 1886, 2819]. In a newly hatched larva (∼24 h AEL), the major discs contain 20–70 cells (wing ∼40; eye ∼70; leg ∼40; haltere ∼20; genital ∼60) [2650]. In mid-to-late 1st instar, all disc cells resume mitosis [474, 528, 2650] (they had stalled at ∼10 h AEL, St. 13 [1142, 1259]) and start multiplying exponentially [3139, 3441]. During the 3rd instar, cell numbers double every ∼10 h (wing 8 h; eye 11 h; leg 8 h; haltere 13 h) [2081, 2935], so that by pupariation each disc has 10–50 × 103 cells (wing ∼49,000; eye ∼44,000; leg ∼17,000; haltere ∼10,000) [542, 1123, 1185, 2710] and measures 50–300 µm across [524, 3139, 3507]. Indeed, one reason for

86

studying discs is that they must pattern themselves during growth – a constraint that is typical of most developing systems (e.g., vertebrates) – whereas ﬂy embryos employ a quirky syncytial phase whose circuitry may be less useful in trying to understand common (ancient) patterning mechanisms [1303, 3074, 3747]. When do disc cells become determined? That is, when do they irrevocably decide to make (1) adult vs. larval structures [1717] and, more narrowly, (2) structures appropriate to their own disc type vs. some other type [523, 1411, 2411, 3139, 3795]? Both decisions must occur after 3 h AEL (St. 5) because single blastoderm cells can contribute to both imaginal and larval tissues when transplanted homotopically at that time [2819]. Leg determination probably happens by 5.5 h AEL (St. 11) because the early enhancer for Distal-less (Dll) is activated then [827, 1571, 1572]. Dll is a marker for distal leg, although its expression is not so restricted initially [1572]. The ﬁrst transcription of vestigial (vg), a marker for wing and haltere cells, is not detected until ∼2 h later [827, 4681]. Strangely, however, neither Dll nor vg is required for disc development at this stage [827, 3969, 4681], so neither gene provides ironclad evidence for determination [884, 4682]. Nevertheless, the ∼5–7 h AEL (St. 11) estimate is probably close [2335], given that (1) disc fates can be changed until that time (but not afterward) by misexpressing homeotic genes [684, 3930] and (2) X-rays can induce pattern duplications in discs (implying that they have become embryonic ﬁelds) after 6 h AEL (but not at 3 h AEL) [4651]. Remarkably, the Vg-expressing cells in Segments T2 and T3 originate as subsets of Dll-expressing cells that move dorsally in the extended germ band [827, 1571, 1572]. Because this movement is correlated with locally increased mitosis [237], it is probably driven by growth, although cell migration is likely involved later when wing and haltere cells cling to tracheal branches [237, 2335]. Evidently, wing and haltere progenitor cells arise within the same nests that form 2nd and 3rd legs, respectively (Fig. 4.4). This conclusion ﬁts with the visible “budding” of prospective wing from 2nd-leg rudiments (and haltere from 3rd leg) in the dipteran Dacus tryoni [81], and it afﬁrms the controversial idea that the insect wing evolved as a branch from the arthropod leg [145, 2348, 2349, 3911, 4662]. Also, it clariﬁes why single blastoderm cells can contribute to both wing and 2nd leg (or to haltere and 3rd leg) [2442, 4076, 4651]. Moreover, this recent ﬁnding settles an old dispute between two titans from the Morgan era: A. H. Sturtevant and Curt Stern. In his 1929 gynandromorph study (cf. Ch. 1), Sturtevant noticed that sternopleu-

IMAGINAL DISCS

ral bristles are clonally linked with thoracic bristles as often as with leg bristles [4180]. From this result, he inferred that the sternopleura (a ﬂank sclerite; cf. Figs. 4.4i and 5.1d) can come from either the wing or 2nd-leg disc, depending on which disc happens to spread into this region ﬁrst during metamorphosis (cf. other claims for indeterminacy [3250] that were refuted by Stern [343, 3006]). Stern argued (correctly) that sternopleurae always come from 2nd-leg discs, and he marshaled much evidence to prove his point [4090, 4099]. Sturtevant remained stubbornly unconvinced [4182]. To explain Sturtevant’s data, Stern guessed that leg and wing primordia must abut one another in the ectoderm [4090, 4652], and Dll and Vg now validate his 1940 conjecture: If the division line between [male and female areas] of a developing gynandromorph has an equal chance of falling either between the ventral [leg] and dorsal [wing] anlage or between the two subregions [sternopleura vs. remainder] of the ventral [leg] anlage, then the results of Sturtevant can be explained without recourse to the hypothesis of indeterminate overgrowth of imaginal disc ectoderm during metamorphosis. [4090]

By ∼7 h AEL (St. 12) when disc fates appear to be set, the leg and wing primordia contain ∼20 and ∼30 cells, respectively [827, 828, 2819] (including mesoderm cells [1886]), and these ﬁgures agree with indirect estimates from cell lineage studies [2828, 3139, 4649], which put the number of prospective epidermal cells per disc in the 10–40 cell range (excluding labial and clypeolabral discs). Because discs are not clones (cf. Ch. 1), the discinitiating factors (whatever they may be) need not be so precise as to pinpoint single cells in the embryonic ectoderm. Discs retain their identities during larval life [2411], although their cells look embryonic and do not differentiate until metamorphosis [82, 526, 3795, 3797]. Thus, it is not possible to distinguish wing- from leg-disc cells, for instance, just by looking at them [3165, 4424]. This prolonged period of “determination sans differentiation” [1654, 3062, 3448] made it possible to study the stability of determined states [1384, 1406, 1668] and growth regulation [543], which led to the discovery of transdetermination and regeneration, respectively [2755, 3794]. The independence of larval from imaginal development is vividly illustrated by discless larvae, which feed and grow and molt apparently normally and only die when attempting to undergo metamorphosis [3428, 3880, 3886, 4221]. Disc cells remain diploid [499, 1201, 1764, 1765, 2151], whereas the larval skin cells that surround them become polytene [1126, 1259, 1346] via endocycles of DNA replication

CHAPTER FOUR. ORIGIN AND GROWTH OF DISCS

[1261, 3551, 3927, 3995, 4587].

Historically, differences like these made sense in terms of the once-popular view that separate batteries of genes control imaginal vs. larval development [1384, 4686].

To my mind the simplest explanation of such a clear cut dichotomy brought about by a single kind of stimulus is to be found in the analogy of the locked door to which the juvenile hormone is the key. In other words we are concerned with two sets of genes so disposed that in the presence of a small amount of juvenile hormone one set takes precedence, while in the presence of a large amount of juvenile hormone the other set is brought into action. [4659]

That view was primarily buttressed by “disc-speciﬁc” mutations recovered in screens for pupal lethals [3880, 3881, 3886, 3888, 4121] (especially ones that cause a discless phenotype), but subsequent work showed that defects can be disc-speciﬁc for a trivial reason -- viz., the egg has enough maternally supplied gene products to build the larva (in 1 day) but not the adult (requiring 4 more days) [834, 4221, 4257, 4671]. Regardless of whether the “Battery Dichotomy Hypothesis” has any validity in other areas (e.g., physiology), it is certainly defunct in the realm of patterning: many of the same genes that pattern the larval epidermis also pattern discs [834, 3341, 4675] and histoblast nests [4158]. Except for the humeral disc (which lacks a lumen), discs become hollow sacs via invagination or delamination [1421, 1544]. The apices of their cells face the lumen [142, 2650, 4424] and rufﬂe to form a carpet of microvilli prior to cuticle secretion [194, 3421, 3422]. The disc epithelium is one-cell thick [3165, 3426, 3539] like the ectoderm whence it comes [1259] and like insect skin in general [2584, 3421, 4661]. One side of each disc thickens into a columnar epithelium that will secrete adult cuticle [1133, 1315, 1431, 3564], while most of the other side forms a thin “peripodial membrane” whose squamous cells make few cuticular parts in the thorax [500, 2862, 2863] but signiﬁcant parts of the head [1777]. Curiously, mitoses may be synchronized across the lumen [2744]. Coordination of this sort (as well as other sorts of signals) is probably mediated by the ﬁlopodia (“translumenal extensions”) that stretch from peripodial cells to the columnar surface [773, 1473, 3508]. During 3rd instar, the columnar epithelium looks “pseudostratiﬁed” because its nuclei occupy many levels [2650, 4715]. In early 3rd instar, the wing portion of the wing disc (as distinct from the notal part) is initiated via a separate genetic circuit [885, 2219, 2254, 4683, 4684]. Characteristic folds emerge there (eventually forming a “pouch”) and in the leg disc (eventually forming a comparable “endknob”) [142, 377, 2287], presumably due to region-speciﬁc mitotic

87

rates or orientations [544, 2848, 3422, 4427] or histotypic afﬁnities [351, 1060, 2570]. Cell death cannot play a major role in morphogenesis or patterning [532, 3422] because it is so rare [12, 1501, 2015, 2848, 2849], except in eyes [396, 397, 489, 1309, 1763] where it serves to tighten the ommatidial lattice [4713, 4715]. Aside from epithelial cells, most discs contain adepithelial cells [239, 476, 497, 3104, 3660] (= mesodermal myoblasts [236, 1214, 1886, 2820, 3609, 3661]), neurons [4330], and tracheal cells – all of which reside between the epithelium and the basal lamina [3422, 3426]. Genetic evidence suggests that adepithelial cells in leg discs induce patterning in the tarsal epithelium [3332, 3333]. The male genital disc is freakish insofar as it recruits mesodermal cells into the epithelium [38]. Those cells go on to form the paragonia and vas deferens. During metamorphosis, each disc everts through its stalk [1311, 3422, 4429]. Historically, the planarity and insularity of discs (the latter feature being uncommon among embryonic ﬁelds [4516]) proved useful for analyzing mechanisms [523, 2429, 3448], as did the durability and intricacy of the cuticle, which indelibly records the ﬁnal state of virtually every epidermal cell [526, 3421, 4663].

Thoracic discs arise at Wingless/Engrailed boundaries Although blastoderm cells are not restricted to larval vs. imaginal fates [2819], nor to ventral (leg) vs. dorsal (wing/haltere), nor even to left vs. right disc fates [4076, 4651], they are conﬁned to anterior vs. posterior fates within each segment [4076]. Why do A/P compartments exist? One early clue was that A/P boundaries precede disc formation, so they might help decide where discs arise [2812, 3135]. In fact, they do. The wing’s A/P border traverses a featureless ﬁeld of hairs (Fig. 4.4) [354, 1376] – a ﬁnding that seemed odd because PI axes were thought to be “seams” for anatomic designs [904, 1409]. This invisible line was previously revealed in the phenotypes of bithorax LOF , postbithorax LOF , and engrailed1 mutants [4671]. The ﬁrst two mutations reside in the cis-regulatory region of Ultrabithorax (Ubx) [254, 3940] and hence are written more correctly as “UbxbxLOF ” and “Ubx pbxLOF ,” whereas en1 is an idiosyncratic allele of engrailed [2356] that does not behave as a simple LOF [852, 1138, 1636, 1837, 2447]. UbxbxLOF and Ubx pbxLOF transform the A or P part of the haltere into the corresponding part of the wing [2924] and A or P 3rd leg into 2nd leg [4324], whereas en1 transforms P into A compartments in the wing [493, 1378, 2306, 2441] and foreleg [439, 852, 4343]. Thus, while the Ubx alleles might be acting via interdisc

88

IMAGINAL DISCS

CHAPTER FOUR. ORIGIN AND GROWTH OF DISCS

serial homologies, en1 cannot be doing so. Nevertheless, all three alleles ﬁt the Selector Gene Hypothesis since they imply an abstract (histotype-independent) code of some sort [1355, 1375]. Because en is on in the P part of each disc and off in the A part [495, 1697, 1963, 2307], it could serve as a PI “mode selector” gene to allow A and P cells to use different interpretation modes for reading the same PI gradient [2441,

89

2930]. If so, then the A and P gradients should be “back to back” [904, 4734], given the mirror-image phenotype of en1 in the wing and leg. Comparable duplications caused by other mutations are attributable to cell death followed by compensatory growth [523, 526, 539, 1499, 2015, 3441], but en1 cannot be acting thusly because it mainly behaves cell autonomously [1378, 2441, 2928, 4343]. Indeed, its autonomy led to Morata and Lawrence to postulate that

FIGURE 4.4. Initiation of thoracic discs at the Wingless/Engrailed interface and the later role of the A/P boundary during disc development. Gene abbreviations: dpp (decapentaplegic), Dll (Distal-less), en (engrailed), hh (hedgehog), vg (vestigial ), wg (wingless). a, b. Magniﬁed view of the embryo’s T2 (second thoracic segment, ﬂank area only) at two stages after egg laying (AEL). Axes (A-P, anterior-posterior; D-V, dorsal-ventral) are indicated by compass at left. a. T2 at ∼5 h AEL (extended germ band, St. 10/11). The posterior two rows of cells (hexagons) transcribe wg or en; key at right) and secrete Wg or Hh. Wg and Hh diffuse (black triangles = concentration gradients) and sustain one another’s synthesis by a feedback circuit [2717] that is abridged here (inset at right; see Fig. 2.7 for key and [2816] for theory). Because the gradients are symmetric, each should create two ﬂanking counterparts, but the response is asymmetric due to other genes (not shown) [596, 1628, 3129, 3748]. Before the stage in b, all ectoderm cells divide at least once. The pedigree (lines connect mother to daughter cells) depicts mitoses in one ﬁle of cells along the A-P axis, although divisions are not really so orderly. The important point is this: whether a cell expresses Wg or En is not heritable. To wit, as cells move away from the Wg/En interface, they stop making En [4491] and Wg [3363] (fading letters). b. T2 at ∼8 h AEL (germ band shortening, St. 12). Wg and En domains do not grow in proportion to segment width (although En stripes widen more than depicted [272, 1064, 2717]) because diffusion ranges of Wg and Hh are ﬁxed [1063]. An A-P stripe of dpp-on cells crosses a gap in the Wg stripe. Dpp is also a morphogen (gradient at right). Around the tip of the Wg stripe remnant, cells transcribe Dll [834]. The upper limit of the Dll-on spot is set (at ∼St. 11) by Dpp (Dpp Dll). The lower limit is set by Spitz (Spitz Dll) – another morphogen (not shown; cf. Fig. 6.12) from the V midline [1572, 2335, 3536]. c–e. Subsequent changes in the T2 Dll cluster. Reshaping is apparently due to mitosis (vs. active cell migration) [827, 1571], but cells also rearrange to some extent [1572, 2335]. c. Note the new symbol key. d. As cells move dorsally with the Dpp stripe (see e), they stop making Dll [1571] (fading letters). e. Eventually a dorsal cohort starts transcribing vg – a wing (and haltere) marker. Thus, wing and leg anlagen arise from a common pool of cells [827]. The pool is partitioned (St. 15) based on how much Dpp (D signal) the cells perceive relative to Spitz (V signal; not shown) [2335]:

High Dpp /Spi ratio Low Dpp /Spi ratio

“wing” state (vg-on/Dll-off) “leg” state (vg-off/Dll-on)

f–i. Mature (3rd-instar) discs and their adult derivatives, showing regions that do (black) or do not (gray) make En (oriented as per compass in a). Within both discs, the en on/off boundary becomes a reference line for specifying cell positions along the A-P axis, using either Wg or Dpp as a morphogen (not shown). In the leg disc, a remnant of wg-on cells is retained ventrally, and a stripe of dpp-on cells is induced dorsally (cf. Fig. 5.4). In the wing disc, a stripe of dpp-on cells is induced along the entire boundary (cf. Fig. 6.3). Plusses mark the appendage tips, which would come out of the page during eversion and then ﬂop down. f. Left wing disc. Tissue in the translucent rectangle (D wing surface) is not visible in g because it folds behind (cf. Fig. 6.1). Eversion brings the notum (NP, PA, and SC = notopleural, postalar, and scutellar areas) into contact with the pleura (pl). g. Adult wing (V surface only) and heminotum in side view. Bristles are omitted except at wing margin. A and P denote lineage compartments. The A edge of the en-on region per se is actually closer to vein III due to a shift during late 3rd instar (cf. Fig. 6.7d) [350]. h. Left 2nd-leg disc. i. Adult leg, drawn as if ﬁlleted along the ventral A/P boundary and spread ﬂat so its whole surface is visible. Thoracic structures also come from the leg disc, including StPl (sternopleural sclerite). Holes in joints are cartographic artifacts due to odd shapes of proximal leg segments. Panel a is based on [1063]; b–e are adapted from [834, 884, 1571]; f is traced from [2467]; g is modiﬁed from [1376, 4076]; h is sketched from stained discs pictured in [495, 2754]; and i is amalgamated from [1800, 2449, 4076]. N.B.: Cells are not usually packed so neatly [1064], nor are they so constant in shape or size [4743]. Indeed, they may not even stay in a monolayer [1572]. In b, dpp transcription is actually greater posteriorly along its stripe (possibly due to upstream control by segment-polarity genes) [2003]. In c–e, a ring of escargot-on cells (not shown; future pleura and coxa) surrounds the Dll-on core (all other leg segments) [1542, 1572, 1573]. Unlike the leg disc whose axes persist from the blastoderm, the wing disc appears to rotate ∼90◦ during development (not shown) [4652]. As shown in h, the A/P (en-on/off) line of the leg disc is diagonal near the center but zigzags toward the stalk above [495, 3747, 4254]. This irregularity has led to confusion because some authors schematize the A/P line vertically (D pole at 12 o’clock) like the outer part, while others slant it (D pole at 1 or 2 o’clock) like the central part (cf. Fig. 5.1).

90

“the state of activity of the selector gene is permanent and . . . propagated by cell heredity” [2440], and the discovery that en has a homeobox [1031, 1247, 3429] corroborated this conjecture [354, 1979]. Subsequent research, however, showed that at certain stages a cell only expresses en as long as its neighbors force it to do so [1839, 1968, 3310, 4491]. In general, the following terms help distinguish between these autonomous (independent) and nonautonomous (dependent) states [523, 3448, 3795, 3796]: 1. “Determined” states are intrinsic, permanent, and heritable [1411, 1421, 2448]. They are “ﬁrm biases” [3794]. 2. “Speciﬁed” states are extrinsic (imposed by intercellular signals), transient, and not heritable [2111, 2716, 4645]. They are “transitory biases” [3794] that depend on the persistence of the external signals. En and Wingless (Wg) stripes ﬁrst appear at the cellular blastoderm stage (∼3 h AEL, St. 5) [67, 176, 271, 2146, 3157]. When a dye was injected into single cells and their descendants were examined after several mitoses (∼5– 8 h AEL), 15 (out of 37) clones were found to contain a mixture of En-expressing and nonexpressing cells [4491]. Clearly, En states are not irrevocably ﬁxed in the blastoderm because if they were, then only pure (en-on or en-off) clones should exist. A second major ﬁnding in this study was that none of the 15 clones straddled a Wg/En (parasegment) boundary [1963]. The inability of blastoderm clones to cross this line conﬁrmed cell-lineage studies of leg and wing discs [2432, 4076] and showed that A/P lineage restrictions apply not only to discs, but also to the ectodermal fabric from which they are cut [2444, 4488]. In theory, segregation of A and P cells could be enforced by killing trespassers, but it seems that cells never even venture into alien territory. The reason appears to be an incompatibility of cell afﬁnities [2437]. That is, the en-on state endows a cell with (as yet unidentiﬁed) surface molecules (cadherins? [3308]) that prevent its immersion in a ﬁeld of wg-on cells, and vice versa. The idea that disc cells seek “their own kind” came from old experiments where cells from marked discs were mixed together, and the aggregates were found to form chimeric patterns. The harmonious integration in these patterns was attributed to homophilic cell sorting [1356, 4423, 4425], but it could equally have been due to repatterning during growth [526, 1357, 1775, 3424, 3448]. To distinguish between these alternatives, short-term cell movements (sans growth) were monitored and disctype preferences were thereby proven [1202, 1205]. A vs. P

IMAGINAL DISCS

preferences had been suspected from the behavior of en1 clones in mosaics [1358], and recent evidence supports this general notion (cf. Ch. 6) [354, 2437, 2993]. From ∼3 to 5 h AEL (St. 5–10), En states are sustained by signals that come from the adjacent Wg stripes (Fig. 4.4) [271, 1093, 2719, 3921, 4489]. Manifestations of this dependence include (1) decay of En expression in cells as they move away from the Wg/En border during segment growth [4491] and (2) premature decay of En stripes in wg null embryos [1065, 2719]. Interestingly, the dependence is mutual [1063, 1836, 1841, 4743]. Wg expression is sustained by a Hedgehog (Hh) signal emitted by the En-expressing cells [1969, 1977, 1982, 2494, 2719]. Wg and Hh are both secreted proteins [334, 574, 1789, 4440, 4455], whose modes of action are discussed later. For now, the essential point is that the diffusion ranges of Wg [271, 920, 1541, 1779, 4442] and Hh [1969, 4262] are a only few cell diameters in the embryo [3748], so any cell that moves outside this range will cease expressing whatever genes were dependent on that morphogen [2810, 2814]. The third morphogen that is instrumental in disc development is Decapentaplegic (Dpp). Cells whose states depend on morphogens can be thought of as revelers in a nightclub. If the colored lights (≈ morphogen ranges) are aimed at ﬁxed spots on the ﬂoor (≈ epithelium), then dancers can jostle (≈ divide or rearrange) into or out of a colored area, but each area will be occupied by roughly the same number of people at any given time. The system is in a steady state, despite a ﬂux of its parts. After ∼5 h AEL (St. 10) both en-expressing cells [1093, 1790, 3129] and wg-expressing cells [1894, 2013, 2534, 2536, 2683] adopt determined states that are analogous to indelible skin colors [2717]. This “Cabaret Metaphor” also pertains to how discs arise. By ∼5 h AEL (when germ band extension is completed), Wg (but not En) stripes split into ventral and dorsal pieces [176, 271, 880, 1093] with an intervening gap (Fig. 4.4). A perpendicular (A-P) stripe of Dppexpressing cells intersects the tips of the ventral (D-V) Wg stripe remnants [2003, 3833], forming a ladder of struts (Dpp) and rungs (Wg) along the trunk [827, 3122]. At each Dpp-Wg intersection in T1–T3, clusters of cells make Dll. This coincidence implied that Dll is only transcribed in cells that receive both signals [827, 833]: {Wg and Dpp}

Dll?

Along the A-P axis, Dll is activated by Wg (see below) and limited by Wg’s range of diffusion. However, dpp null embryos still express Dll [1572], so dpp cannot be needed for activation. On the contrary, Dpp must be

CHAPTER FOUR. ORIGIN AND GROWTH OF DISCS

stiﬂing Dll dorsally because Dll spots elongate dorsally in dpp null embryos [1572] (cf. similar effects on salivary gland anlagen [1820]). Ventrally, Dll is conﬁned by Spitz, another secreted signal [1572, 2335]. Together, Dpp and Spitz conﬁne Dll along the D-V axis. Thus, the actual rule is {Wg and NOT-Dpp and NOT-Spitz}

Dll.

Still unexplained is how Dll can coexist with Dpp at Dpp-Wg intersections if Dpp inhibits Dll. Conceivably, some factor (Wg?) overrides Dpp’s inhibition of Dll at those points. In each thoracic segment (of wild-type embryos), the Dll clusters straddle the Wg/En border [1571]. Hence, both states (Wg and En) get incorporated into the founder population of prospective disc cells. As described above, these cells are not yet segregated into ventral (leg) vs. dorsal (wing/haltere) fates. Soon thereafter, subgroups split off (under the inﬂuence of Dpp [1572]) and migrate dorsally (tracking the Dpp stripe [1571]). Dll expression wanes in the cells as they move away from the Wg rung tips [827, 1571], so Wg is probably sustaining Dll expression (the Cabaret Scenario). Dependence of Dll on Wg was conﬁrmed by showing that Dll disappears when Wg is artiﬁcially inactivated (by heat-pulsing a t.s. mutant) at this stage [827]. The turning on of the winghaltere marker Vg in the dorsally migrating subgroups in T2 and T3 [1571, 1572] may require that Dll be turned off. That is, while Dll confers the potency to make Vg, such “competent” cells can only make Vg when they subsequently shut off Dll. This “ON-then-OFF Licensing Trick” is also used elsewhere (e.g., Dll-Hth’s launching of spineless expression in the antenna, cf. Fig. 8.3). Leg vs. wing states are imprinted at ∼6 h AEL (St. 11) by the relative doses of dorsal (Dpp) and ventral (Spitz) signals that the cells perceive [2335]. Inside the nascent discs, cells cannot be undergoing any drastic rearrangements because their blastodermal fate maps roughly match the layout of adult structures [3602, 4652]. Nevertheless, leg cells must reorganize to some extent because the future proximal cells are initially located dorsal to the future distal cells, whereas they later surround them. This situation arises because these fates are assigned by differing intensities of Dpp signal [1572]. Wing disc initiation is also more complicated (in a genetic, not a topological, sense). It involves two phases. In Phase 1, Vg (a transcriptional co-activator [1686]) is expressed (as described above) together with Escargot (Esg) and Snail (Sna) in response to extrinsic inductive cues (“{Dpp and NOT-Wg} Vg”?). Esg and Sna

91

are zinc-ﬁnger proteins whose preferences for DNA sequences are virtually identical, and so are their downstream effects (as deduced genetically) – a redundancy remarkably like the bHLH proteins Achaete and Scute [1333]. In Phase 2, Esg and Sna establish an intrinsic “wing” or “haltere” state by auto- and cross-activation (again like Ac and Sc), and Vg then comes under their control [1333]. The above scenarios do not apply to genital [679, 735, 1170, 2028, 3817] or eye discs [2933, 3041, 4146], each of which comes from several segments [2103, 4825]. Indeed, the eye disc does not acquire an A/P boundary until long after it invaginates from the ectoderm (i.e., during 2nd instar) [1635, 1637].

Cell lineage within compartments is indeterminate Nowhere is the Cabaret Metaphor more apt than within each compartment as a disc grows. Cells are free to jostle relative to the adult regions (colored areas) that they will eventually occupy (when the music stops) [2142, 3947]. This ﬂuidity was revealed by Sturtevant’s studies of gynandromorphs [4180] (cf. Ch. 1), and it was conﬁrmed at a ﬁne-grained level in all major discs by randomly marking cells at various stages of development [3441]. Because descendants of marked cells tend to stay together [3441], most cell movements must be due to passive displacements [3, 1610, 1888, 1890] (newborn cells pushing extant ones? [1525, 3515, 3518]), rather than to active migration, which would fragment the clone [532, 3422, 4671]. Clones tend to have irregular outlines [3441], and when outlines from different individuals are superimposed, they tend to overlap in virtually every region of the body [532], including the eye [189, 260, 261], antenna [3446], leg [544], wing [521, 1545], notum [521, 3007], and genitalia [1107]. The only exceptions are the compartment borders. The ﬂexibility of cell lineage is epitomized by Minute mosaics [354]. When single cells are spurred to grow faster than their neighbors (by expulsion of a retarding Minute LOF allele), their descendants can occupy more than 10 times the area that they normally would within the A or P compartment without disturbing the pattern [497, 2935]. One consequence of this plasticity is robustness [543, 2375, 2448]. Amazingly, 75% of a disc’s cells can be killed without incurring any cuticular defects [1776]. The dead cells are simply replaced by compensatory growth. Apparently, the goal of disc growth is to generate enough tissue to ﬁll all subregions, regardless of cell pedigrees: “the developing pattern seems to control proliferation, rather than the other way around” [532].

92

Strictly speaking, it is not proliferation per se that is being controlled, but rather tissue mass [4579]. Cell sizes can be altered drastically without affecting disc size, gene expression zones, or cuticular pattern [2081, 2771, 3081, 3750, 4576]. Any PI gradients that exist in discs must therefore be heedless of cell boundaries [546, 3862]. In other words, the “graininess” [1822, 2016] that cells impose on the overall “image” is irrelevant to the production of the image itself. Disc cell proliferation is sustained by circulating growth factors from the larval fat body [2707] and possibly the brain [1346]. Endocrine signals like these are transduced by the insulin receptor pathway [486, 3450] , which responds to insulin in cultured cells [919] and has significant effects on cell and organ size in vivo [2476] . Within the leg and wing discs, the chief paracrine stimulant appears to be Dpp [569, 619, 643, 1674, 1839], and the same is true for the eye disc during its early stages [570]. Nevertheless, many other pathways participate [543, 1126, 1142, 4667, 4886], including dActivin [511] , Hh [2214] , Wg [734] (although, oddly, not in the wing pouch [2254] ), EGFR [3470] (including the wing pouch [3025] and eye [185] ), JAK-STAT [1906] , and Notch [2362] . Indeed, Dpp can only elicit outgrowth of the leg [617, 4490] or wing [558, 559, 617, 643, 3862] by cooperating with an unknown factor whose expression and regulation mimic those of Wg [2254] (cf. Chs. 5 and 6). A common conduit for multiple growth-regulating pathways is the Ras-MAPK cascade [2132] , which promotes dMyc stability [1342, 2081, 3846, 4124] and blocks apoptosis [2793] . Discs also use anti-mitogenic signals that evidently rely on junctions [533, 1907, 4745] and cadherin-like adhesive proteins [1386] because LOF defects in these components can cause overgrowth [4742] . One such retardant is nitric oxide [2370]. There must not be many such restraints on growth, however, because no single or compound LOF mutants have ever been found that rival the drosophilid titans of Hawaii [669]. Indeed, no such giants have yet been produced by any GOF manipulations either [2561]. Until about mid-3rd instar, mitoses are distributed evenly in leg discs [1599], and the same is true for wing discs [20, 2015, 2848, 4427]. The uniformity is attributable to a compact (≤0.7 kb) cis-enhancer that ensures ubiquitous expression of String – a rate-limiting (Cdc25) regulator of mitosis [2480]. Around mid-3rd instar, there is a transition from random to patterned mitoses. The change is probably due to a switch from the previous (cell-size responsive?) enhancer to other (prepattern responsive?) enhancers at the string locus [2480]. Proneural areas be-

IMAGINAL DISCS

come quiescent [1925], including the future wing margin [2079, 2081, 3154, 3374]. So does a band of cells just anterior to the A/P compartment boundary [543, 3813] (possibly dpp-on cells [2739]) and a spot at the center of the leg disc [1599, 4666] (possibly aristaless-on cells [620, 2287]). In the early pupal period, most SOPs undergo their differentiative divisions, and mitoses occur in a belt around the notum, along wing veins, and then in the interveins [1545, 1741, 3813]. Proliferation ceases one day after pupariation [1312]. Cessation of growth must be controlled intrinsically because wild-type discs do not exceed their normal size when given extra time to grow (via pupariation delay [3955] or transplantation [19, 542, 546, 1357]). In theory, terminal size could be targeted in terms of total cell number [99, 480, 1548, 3483, 3492], but in fact, as stated above, growth is instead shut off at a certain mass [2078, 3081, 4576]. The unresolved “Growth Cessation Mystery” is: what local cues do cells use to monitor such a global property [3416, 4114, 4196, 4886]? One possible answer is that some morphogens also act as growth (or survival) factors [847, 3862]. In that case, organ dimensions could be limited by the range [855, 2291, 4265] and/or slope [1142, 2448, 2852] of a gradient that is steeper in young discs [977, 1169, 3213, 3862]. Indeed, discs with extra Dpp-producing zones can triple in breadth [4229, 4418]. Whatever the mechanism, it evidently operates separately in different compartments because their sizes can be manipulated independently in the same disc [1169, 3947, 3961, 4265].

The Polar Coordinate Model linked regeneration to development When mature discs are cut into pieces and forced to metamorphose, each piece forms a certain subset of elements. In this way, fate maps were charted [526, 3165]. In 1971, experiments were described where parts of leg discs were allowed to grow before metamorphosis. Regardless of whether postsurgical growth occurred in situ [522] or in an adult host [3808], the upper (stalk-end) half typically regenerated (i.e., made a whole leg), while the lower half “duplicated” (i.e., made its fated structures but in two copies). This outcome can be restated as “ABC makes ABCDEF, and DEF makes FEDDEF,” where letters are identities (∼ dorsal to ventral) and underlined elements are added postsurgically. Lateral and medial halves also obey this “Reciprocity” Rule [3808]). That is, one fragment (medial) regenerates while its complement (lateral) duplicates.

CHAPTER FOUR. ORIGIN AND GROWTH OF DISCS

To explain why leg discs behave in this way, Peter Bryant proposed that cells at a cut edge can only adopt lower values in a PI-like gradient [522, 523]. If 654321 denotes the gradient (each digit = 1/6 of the disc along an axis), then this “Down the Slope” constraint means that both halves must make DEF because they share the same (∼3.5) wound edge [20]: 654 (ABC) makes 654321, and 321 (DEF) makes 123321. This “Gradient of Developmental Capacity (GDC) Model” argues that a cell’s positional value dictates its regenerative repertoire [525]. Research soon shifted to the wing disc because its larger size offered much higher resolution. Bryant bisected wing discs at 5 longitudinal and 8 transverse levels, and found that the fragment pairs obey the same Reciprocity Rule as leg discs [524, 525]. These orthogonal series suggested a Cartesian coordinate system, but diagonal cuts behaved similarly. Thus, regeneration proceeds in all directions away from a peak near the center of the disc, indicating a cone-shaped gradient (Fig. 4.5c). Tests of the model, however, produced confounding results: 1. Paradox 1 (Fig. 4.5d): the central fragments. Any piece that contains the high point should regenerate, but central pieces (bearing the gradient’s peak) actually duplicate [536]. Bryant initially guessed that loss of the peripodial membrane from these 4-cut (“cookie cutter”) fragments might be responsible for this odd result [524]. 2. Paradox 2 (Fig. 4.6a): the quadrants. The regenerative power of a fragment should only depend on its wound edge(s). However, all four quadrants duplicate, and complementary pieces regenerate, so the behavior of an edge actually seems to depend on whether it belongs to a 1/4 or 3/4 piece [526]. 3. Paradox 3 (Fig. 4.6b): intercalary regeneration. Because the gradient’s peak is central, marginal pieces must duplicate. When such pieces are cultured singly or intermixed with identical fragments this result is indeed observed. However, when pieces from opposite sides of the disc are co-cultured as a scrambled mass, they regenerate the midsection, including the peak [1773, 1775]. Intercalary regeneration had been previously documented in cockroach legs [386, 539], and the Reciprocity Rule had been found to govern amphibian limbs [20]. A concerted effort (among Peter Bryant, Susan Bryant, and Vernon French) to reconcile these diverse systems led to the “Polar Coordinate (PC) Model” in 1976 [1303].

93

In contrast to the GDC Model, the PC Model explains the Reciprocity Rule by postulating that growth depends on interactions between wound edges, rather than on the free edges themselves [354, 536]. It departs from Wolpert’s idea of morphogen gradients insofar as it relies exclusively on communication between adjacent cells [4682]. Cells are presumed to execute only two cardinal instructions: 1. “Shortest Intercalation” Rule. When cells are confronted at wound edges, they assess one another’s coordinates. Disparities provoke mitosis, and newborn cells adopt values between those of the ﬂanking cells. In so doing, the cells pursue the shorter of the two possible arcs (on a circle) that connect the points. How cells might compute or compare alternate path lengths was never clear [1805, 2291, 2808, 3862]. Because the intercalated span of values is identical for complementary fragments, only the context dictates whether the new growth is “regeneration” or “duplication,” but this distinction is moot to the cells themselves. Typically, the model is illustrated as a clock face, but cells are not supposed to perceive the “12/0” meridian as a discontinuity. Hence, the PC system is seamless, except for a singularity at the origin where opposite angular values converge like a contracted purse string. What happens to the rules at this point (the “Central Degeneracy Problem”) was never solved [3915, 3916, 4698]. 2. “Distalization” Rule. In the original version of the model, a complete circle of circumferential values was thought to be essential for newborn cells to “distalize” – i.e., adopt fates at the next level (ring) in a distal (central) direction. (How cells sense completeness was unclear.) Exceptions [3811, 4141] led to a revision [547]. Newborn cells were now supposed to distalize whenever their intended coordinates are already taken by cells at that radial level (regardless of whether a whole circle is present). The revised Rule 2 conforms with Rule 1 insofar as it also depends solely on nearest-neighbor interactions. The geometry advocated by the PC Model was not new (Driesch had toyed with polar coordinates in 1894 [1101, 3742]), but the synthesis that it achieved was historic because it deftly explained a huge mass of confusing data. Also, it was the ﬁrst real hint that arthropods and vertebrates might build their limbs in similar ways. Over the ensuing decades, the model’s popularity waxed and waned [354, 531, 863, 2420]. One lemma, at least, endures to this day: when the normal chain of events is diverted by

94

IMAGINAL DISCS

a

e map fat II

b

III IV

ions ect s i b E F

D 8 7

Key:

B

G

V

fragment duplicates

C

fragment regenerates

6A 5

I

c

4 3.5 3

AP

2

GDC Model

PA

NP

1

V

PS

A

SA

SC

4

F

P

peak

C peak

D

0

2

DC

Paradox

d

FIGURE 4.5. Regenerative potency of wing-disc fragments and an early model.

a. Fate map (abridged) of a mature right wing disc (notum, light shading; arc = suture; wing, dark shading) as per Bryant except that dots are actual SOP sites [1925] and thick lines are prevein zones (I–V) [4189]. Vein I is the bristled part of the margin [1741] (thin dashed line = bristleless part). Thick dashed line is the A/P compartment boundary (cf. Fig. 4.4). Directions (A, anterior; P, posterior; D, dorsal; V, ventral) are given in the compass at right. In all bristle pairs except scutellars (SC; bounded area = scutellum), the P partner is on the right (cf. Fig. 3.4 for abbreviations). White trapezoid is a unique spot (cf. b). b. Results of bisecting a disc lengthwise (B–F) or transversely (1–7) along folds (dashed lines) or elsewhere and letting each piece grow in isolation inside the body of a host adult [524]. As indicated in the key, an unﬁlled arrowhead means that the fragment to its rear “duplicates” (i.e., makes two copies of its fated structures as mirror images), whereas a solid arrowhead means that the fragment behind it “regenerates” (i.e., restores the whole). Note that every cut line has arrowheads of opposite type. Thus, the wing disc obeys a “Reciprocity Rule”: when one piece regenerates, the reciprocal fragment duplicates. The fact that regeneration proceeds away from D, E, 3, and 3.5 lines suggests that the disc uses x-y coordinates, but diagonal cuts (not drawn) showed that potency actually declines radially from the center piece (black trapezoid). c. The Gradient of Developmental Capacity (GDC) Model was based on the ﬁndings shown in b [524]. It proposed that the center piece contains the peak of a conical gradient of regenerative potency. The gradient is schematized here as triangles (side view) above the columnar epithelium (inscribed with imaginary contour lines). When the disc is cut, the gradient is supposed to force cells at each edge to grow down its slope. d. According to the GDC Model, any piece that includes the peak should regenerate, but the “CF24” piece actually duplicates (as does BF16; not shown) [526]. This and other paradoxes (cf. Fig. 4.6) led to the GDC model being abandoned in favor of the Polar Coordinate Model [1775]. [524],

miscues, it can produce global deformities (e.g., trefoil limbs [539, 1805]) that appear freakish to human observers but look natural to the cells themselves because no local rules are broken. The PC Model was theoretically able to encompass normal development as well as regeneration because disc growth in situ might also be regulated by intercalation [1301, 1303]. If nascent discs contain only a subset

of a mature disc’s positional value spectrum – say “1” and “5” of a ﬁnal “12345” array – then juxtaposition of 1 and 5 could lead to birth of a 3, and growth would stop with insertion of 2 and 4. PI (and pattern) would thus be elaborated steadily through such “intercellular negotiation” [834, 4682]. Growth is probably not limited in this way, however, since ﬁnal disc mass is independent of cell density [4576].

CHAPTER FOUR. ORIGIN AND GROWTH OF DISCS

a

D-E

95

Paradox

α

b

β

Paradox

8

8 6

6

3.25

cultured separately

cultured together

2

2

0

δ

11

11

9 10

10 9 9

h e als c inter

d

11 12/0

10 9

7

12/0

1 2 3

interc

4

8 7

6

heals & 9 intercalates 3 4 8 7 6 5

5

6

2

Polar Coordinate Model

alates

10

& alate s

11 12/0

10

4

8

c 12/0

11 12/0 1

11 12/0 1 10 ab 2 c d e 3 9

5

s & lates a

11 12/0 1

1

he inter al c

γ

0

2 3

9

4

8 7

6

5

10

2 3

9

4

8 7

6

5

FIGURE 4.6. Paradoxes that led to the Polar Coordinate Model (cf. Fig. 4.5 for symbol key and context).

a. According to the GDC Model, a fragment’s decision to regenerate vs. duplicate should depend only on its edges, but quadrants (cornered at the inferred peak of regenerative potency) violate this axiom. For example, the α quadrant duplicates, but a piece with the same vertical edge (3/4 complement to β) regenerates. b. Dorsal (“02”) fragments only make dorsal structures, and ventral (“68”) fragments only make ventral ones. However, when 02 and 68 pieces are scrambled together, they regenerate middle (“26”) structures. (See Fig. 4.5 for the numbering scheme.) This result is also hard to explain in terms of free edges. c. The Polar Coordinate (PC) Model avoided both these paradoxes. Based on the phenomenon of intercalation (b), this model argued that growth depends on interactions between wound edges, not on free edges. Every cell is supposed to have a circumferential (clockface) coordinate (= angle from a 12/0 reference line) and a radial (letter) coordinate (= distance from center). The “Shortest Intercalation Rule” asserts that when cells touch (via healing), they assess each other’s coordinates and ﬁll in missing values by the shorter route (no discontinuity at 12/0). For example (at right; cf. b), when “10” abuts “8,” a “9” ﬁlls the gap, instead of “11, 12/0, 1. . . 7” (the longer route). Prime marks denote new values. d. The PC Model obeys the Reciprocity Rule (see α in a). For example, when a fragment contorts to heal “9” and “12/0” edges together, this contact creates “10” and “11” (black tissue), regardless of whether the edges belong to a quadrant (left) or its 3/4 partner (right). Thus, quadrants duplicate because none has enough angular values (i.e., ≥ half ) to regenerate. The various panels are adapted from [524--526,1775].

The idea of a common cellular logic for development and regeneration was supported by (1) the ability of immature disc tissues to provoke regeneration in mature disc fragments [1183], and (2) the ability of cell-

killing agents (e.g., γ-rays [3440], X-rays [3449], UV-rays [1424], or microcautery [425]) to evoke the same kinds of mirrorimage duplications from developing discs as are obtained with fragments of mature discs. In the latter case,

96

the presumption was that necrosis-induced duplications arise via the same route as surgical tissue removal [539]. Indeed, wing-disc structures that maximally resist γ-ray-induced duplication were mapped to precisely the same spot as the GDC cone peak that was localized via surgery (Fig. 6.1) [3440]. Subsequent studies conﬁrmed some of the model’s assumptions, including the topology of wound healing [537, 947, 3564, 3565], the need for contact between cut edges [1184], and the conﬁnement of growth to a blastema at the wound interface [5, 489, 537, 946, 3155]. De novo formation of wing margin between dorsal- and ventral-type cells validated the concept of intercalation [354, 1038], and the ability of different discs to trigger intercalation inter se in a position-speciﬁc manner [18, 535, 1774, 4665] afﬁrmed the idea of a universal PI “language” [3448].

But regeneration has peculiarities that set it apart Contrary evidence, however, also accumulated. Below is a partial list of ﬁndings that deﬁed the PC scheme (see [1301, 2161] for critiques): 1. Violations of the Reciprocity Rule (e.g., fragments regenerating that should only duplicate) were found after tissue maceration [3788, 4141], during long-term (≥1 week) culture [1125, 2144, 2230], and as a function of the fragment’s proximity to – or straddling of [2139, 2143] – the A/P compartment boundary [2140, 2141]. 2. Complementary fragments should grow equally because they intercalate alike. However, duplicating fragments hardly grow at all despite adding more bristles [3808]: they neither double in mass nor grow as much as their regenerating partners [19, 20]. These heretical results challenge the dogma [525] that discs only use “epimorphosis” (patterning that re-

IMAGINAL DISCS

quires mitosis) vs. “morphallaxis” (repatterning sans growth) [2945, 4724, 4736]. 3. The eye-antenna disc should have the coordinate system of a single disc because one part (eye) regenerates and the other (antenna) duplicates [1404, 1406, 1423, 3810]. However, in other contexts (homeosis [3439, 3443, 3447], transdetermination [3788], or ubiquitous Antp expression [1416]) it behaves like a compound legand-wing disc. This “One Disc or Two? Paradox” [1805, 2933, 3788] seemed to have been settled in favor of two discs in 1997 based on the homeotic effects of Distalless [1561], but the one-disc idea rallied in 2000 based on the homeotic effects of the Iroquois Complex [697]. The rule that “antenna never regenerates” [1404, 1411, 1423, 3810] is broken when antennal tissue is macerated before culturing: in ∼20% of the implants, structures form that normally come from the eye part of the disc (ocelli, ptilinum, frons, and head bristles, but not facets) [3788]. Such regeneration should be impossible if the antenna has fewer than half the angular positional values. 4. Blastemas can form at both edges of a fragment [2213] and can regenerate structures in a correct circumferential sequence (as per the fate map) [2144] before healing together. This behavior is more consistent with the GDC Model [523] than with the PC Model. One challenge that the PC Model initially seemed to deﬂect was the sequestering of regenerative potential in a single quadrant of the leg disc [3808]. As explained in Ch. 5, the model attributed this anomaly to a clustering of angular values [1303, 4140], but a recent molecular analysis of wound healing [1472] reveals trans-lumenal interactions that have mortally wounded the PC Model itself.

CHAPTER FIVE

The Leg Disc

In an article entitled “Pattern formation in the embryo and imaginal discs of Drosophila: What are the links?” [4675], Adam Wilkins and David Gubb posed a question that was on the minds of many researchers at that time (1991). The embryo’s segmentation hierarchy was basically understood, but it was unclear what these various genes might be doing in discs [4682]. Wilkins and Gubb argued that segment-polarity genes supply the angular values of the Polar Coordinate (PC) Model, which until then had only been an abstract formalism. Several predictions of this “Angular Values Conjecture” were soon put to the test by molecular genetics. Chief among them was the expectation that LOF and GOF alterations in segment-polarity genes should reorganize disc anatomy. This prophecy was indeed fulﬁlled, and the experimental probing uncovered a trove of insights into the machinery of disc patterning. The conjecture itself, however, turned out to be wrong. Segment-polarity genes do not paint a pinwheel on each disc. Rather, they draw a few important lines – the compartment boundaries.

The Molecular Epoch of disc research was launched in 1991 The Wilkins-Gubb paper, with its clarion call for a molecular assault on discs, provides a convenient demarcation between the Cellular and Molecular Epochs of disc research. In the 1990s, many links were patiently forged between the blastoderm and the adult. During this process, several old puzzles at the cellular level were solved by clever experiments at the molecular level. Among those puzzles were the following [1807]:

1. “Gaps Mystery” (Fig. 5.1e). Cellular Problem: Each leg’s bristle pattern is roughly symmetric about the D-V plane [1883], as are afferent projections of the bristle neurons [3005]. It therefore came as a surprise when the A/P compartment boundary was found not to coincide with this plane, but rather to be offset from it by several cell diameters [1800, 2449]. Shouldn’t they be congruent if this line is used as a reference axis for specifying mirror-symmetric positional information [904]? Molecular solution: The reference axis turns out not to be the A/P boundary itself but a morphogen-producing zone just anterior to it [1807]. There are no gaps between that zone and the mirror plane. 2. “Quadrant Regeneration Mystery” (Fig. 5.2a). Cellular Problem: In contrast to the wing disc, where every quadrant duplicates, one quadrant of the leg disc regenerates [3808, 4140]. Why is this upper medial (UM) quadrant special? Molecular solution: Its peripodial membrane turns out to have a spot of gene expression that dictates regeneration vs. duplication [1472]. The dogma that regeneration and development always use the same rules is false. 3. “Dorsal Remnant Mystery” (Fig. 5.2b). Cellular Problem: When embryos are exposed to X-rays [3449] or UV-rays [1424] at doses that are sufﬁcient to kill cells [2774], the legs that manage to complete development often manifest a “duplication-deﬁcient” anatomy, where part is missing and the rest is duplicated in mirror image. Similar defects can be produced by microcautery [425], by heating t.s. cell-lethal mutants [101, 2102, 3702, 3964], and by in situ excisions [522]. First legs tend to fuse rather than duplicate [3442] because their

97

98

discs are conjoined [377, 3426], but regardless of how the symmetry arises, the remnants tend to retain dorsal structures at the expense of ventral ones [425, 2102, 3442, 3449, 3705]. Why is the dorsal region (outside the UM quadrant) more robust? Molecular solution: It is the source of a diffusible growth factor that stimulates mitoses throughout the disc [529, 1807], and this growth factor is upregulated in response to trauma [2856]. 4. “Triplications Mystery” (Fig. 5.2c). Cellular Problem: Triplications can also be induced in cell-lethal

IMAGINAL DISCS

mutants during certain sensitive periods [1495, 2102]. Typically, such legs have their normal tip (stem) plus an outgrowth (branch) that either diverges or converges (i.e., gains or loses circumferential elements distally). Outgrowths that branch from the stem’s ventral face tend to diverge, while dorsal ones tend to converge [1495] – a bizarre trend also documented in cockroach legs [384, 547, 2804]. Why? Molecular solution: The deciding factor appears to be the mixture of morphogens on one side of the disc vs. the other that is created under these conditions [2856].

CHAPTER FIVE. THE LEG DISC

99

FIGURE 5.1. Geometry of leg development.

a. Mature, left 2nd-leg disc (∼150 µm across [3140]). Lines are folds. The central circle is the “endknob.” Connections to larval skin (stalk) and CNS (nerve) have been cut. b. Longitudinal section (“BM” and “PM” = basement and peripodial membranes; “CE” = columnar epithelium). Transverse lines are cell boundaries. The black cell is one of several larval neurons that innervate Keilin’s organ [834, 2287]. These neurons invaginate with the disc (a piggybacking that is ancient in dipterans [2824]). Their dendrites (not shown) stay attached to larval skin through the stalk [4330], while their axons project to CNS (a route later taken by adult axons). Adepithelial cells are omitted. c. Idealized fate map of left 2nd-leg disc. Segment primordia are the coxa (Co), trochanter (Tro), femur (Fem), tibia (Tib), basitarsus (Ba), and tarsal segments 2–5 (unmarked), with claws mapping centrally. The sternopleura (Stpl) is part of the body wall (cf. Fig. 4.4). AC and PC (shaded) are A and P compartments. Spokes denote bristle rows, whose spacing is estimated: it may not be so uniform (cf. e). As shown below for the tarsus, the annuli telescope out during evagination (toward the viewer) as the covering PM (not shown; cf. b) peels away [1311]. In this schematic, the tarsus also rolls clockwise ∼90◦ . Compass directions (D, V, A, P) refer to prospective or actual adult axes (cf. d, e). d. Anterior face of left 2nd leg (∼2 mm long). There are 2 macrochaetes (distal tibia) and numerous chemosensory (curved) bristles on the tibia and tarsus. “EB” (edge bristle) is at the D midline. Tarsal rows 5–8 are marked on the compass below. e. Basitarsus (whole surface) drawn as if slit along D midline and spread ﬂat. Most bristles have a bract (triangle) and align in rows (numbers at top). Five chemosensory bristles lack bracts and reside between rows, as do 3 sensilla (circles at 3.5, 5.5, and 8.5). Between rows 1 and 8 is a lawn of hairs. Row-1 and -8 bristles are peg shaped and darker; others are tapered and lighter. Aside from bristle thickness and pigmentation, other useful landmarks include bristle lengths and intervals – both of which increase linearly from V to D (graph at top). Indeed, the symmetry is so precise [1883] that it was surprising when the A/P compartments were found to be offset from this D-V mirror plane by a few cells (“gaps” below) [1800, 2449]. Row-1 bristles can reside in either AC or PC (cf. Fig. 3.9c). Panels a and b were traced from [377] and [3426], respectively (see [1774, 2144] for nerve-stalk asymmetry, [1516, 2287] for folding details, and [1311] for region-speciﬁc cell shapes); c is adapted from [1807, 3807]; and e is based on data in [1801, 1803]. N.B.: For reasons explained in Fig. 4.4h legend, some authors orient the axes (c) with the D pole pointing into the stalk (at 12 o’clock vs. ∼1 o’clock here). Claws (c) actually map more dorsally [1812, 3807, 4140]. Numbering of rows (e) follows original nomenclature [1714]. The numbers are backward in [837, 1039, 2533, 4159].

Before examining how these and other riddles were demystiﬁed, it may help to review some of the clues that accumulated before the Molecular Epoch. Those clues tantalized two generations of researchers, and the older analyses were at least as interesting as the studies of the recent past.

Bateson’s Rule (1894) governs symmetry planes in branched legs The latter three mysteries listed above are interrelated insofar as they all involve mirror-symmetric partial patterns. Near the end of the nineteenth century, William Bateson collected numerous examples of such abnormalities as naturally occurring “sports” in various species of arthropods [240]. In virtually every 3-tipped leg, he found that (1) the three tarsi lie in one plane (vs. a tripod), and (2) the outer two are mirror images of the inner one [2809]. These constraints constitute “Bateson’s Rule.” Although the tips always manifest R-L-R or L-R-L symmetry (R and L = right vs. left-handed), the inner member can face the ﬂanking ones with any surface (D, V, A, or P). To illustrate this rule, Bateson built a cute wooden model (Fig. 153 in his 1894 tome) where the bases of three tarsi are geared together. When the in-

ner one rotates clockwise, the outer ones turn counterclockwise, thus maintaining mirror symmetry. Bateson’s Rule must reﬂect a deep law of cellular “psychology” because the syndrome that it describes transcends the mode of tissue removal (irradiation, surgery, or apoptosis) [547]. The PC Model invoked a cellular imperative to “Always maintain continuity!”: when distant cells are apposed, they multiply to ﬁll the gap in positional values by the shortest possible route [539, 2513]. With this basic edict and one ad hoc assumption, the PC model could explain why the UM quadrant regenerates (Fig. 5.3b). To wit, this quarter might contain >50% of the disc’s angular coordinates [1303, 4140]. However, the idea of crowded coordinates undermined the model’s other proposal that discs stop growing when a certain density of coordinates is reached (like people ﬁlling seats in a stadium) [546, 1303]. The PC Model was also able to account for duplications, triplications, and other outgrowths [538, 1496]. Those conjectures were supported by clonal analyses [1497, 1503, 1504, 2102], apoptosis studies [797, 1501], and phenocopy simulations [1498]. In short, the model was robust. Even so, there were doubts. Some of arguments that were contrived to defend the model strained credulity

100

IMAGINAL DISCS

FIGURE 5.2. Mysteries of leg development (cf. text and Fig. 5.1e).

a. Quadrant Regeneration Mystery. Unlike the wing disc, where every quadrant duplicates, the upper medial (UM) quadrant of the 1st leg disc regenerates (black triangles; cf. key in Fig. 4.5). UM regeneration is seen either with or without the endknob (center region) [3808, 4140]. b. Dorsal Remnant Mystery. When cells are killed by X-rays [3449] or cell-lethal mutations [2102, 3705], D structures tend to remain, whereas V ones are lost (except near branch tips). First-leg discs, which are conjoined [3426], fuse medially instead of duplicating, and losses are symmetrical on left (L) vs. right (R). In this contour map, darker zones denote higher frequencies (0–30; –60; –90; –100%) of elements present in 65 pairs of fused 1st legs. c. Triplications Mystery. Triplicated legs possess a “stem” [1875] and a symmetric outgrowth. V-side outgrowths tend to diverge, whereas D-side ones converge, and the (2/6.5) line between cohorts is sharp. Maps in a, b, and c are redrawn from [3808], [3442], and [1495], respectively. N.B.: “Medial” and “lateral” mean toward or away from the larval midline, whereas A, P, D, and V refer to prospective or actual directions in the adult.

because they demanded odd regions of cell loss and contorted angles of healing (Fig. 5.3c–e) [1495, 3442]. Moreover, much of the later data came from a cell-lethal mutation [1502, 3945] whose gene product was then unknown. (Suppressor of forked turned out to be a polyadenylation protein that affects mRNA stability [2879, 4235] and licenses metaphase [140, 141].) Skeptics felt that the model relied too heavily on ad hoc fantasies and molecular phantoms [863, 2161].

Meinhardt’s Boundary Model deftly explained Bateson’s Rule In 1980 (four years after the PC Model’s debut), Hans Meinhardt, a mathematically gifted theoretician, offered a clever new solution to Bateson’s Rule (Fig. 5.3) [2804]. His “Boundary Model” was based on the leg’s A/P compartment boundary and a supposed D/V bound-

ary that subdivides the A region into D and V parts [4076]. Meinhardt inferred that distal tips grow out wherever P, V, and D areas convene [2807]. Normally, this condition is only met at the distal tip, but extra tip “organizers” could arise if P, V, or D cells were to appear in the wrong place due to wounding or apoptosis. The idea that trauma could trigger ectopic gene expression seemed farfetched at the time, but it has since been conﬁrmed [489, 1472, 2856]. As for how three territories might cooperate to induce distalization, Meinhardt conjectured that each cell type secretes an ingredient needed for synthesis of a tip morphogen “T.” That is, the P, V, and D molecules react chemically to make T. Each compartment may be responsible for a particular step in the synthesis of the morphogen or . . . may produce a diffusible cofactor which is required for morphogen production. [2804]

CHAPTER FIVE. THE LEG DISC

The ﬁctional P, V, and D molecules should not diffuse far beyond their source sectors since, in that case, tips would form everywhere. The model thus invokes three short-range inducers and one long-range morphogen. T forms a cone-shaped gradient that could encode radial distance like the PC system’s radial coordinate [2805, 2813]. In Meinhardt’s original model (1980), there is no cellular imperative about continuity. Rather, Bateson’s Rule is an emergent property of the model’s criterion for distal outgrowth [617, 2706]. The geometry is easy to visualize (Fig. 5.3g–i) [2808, 2809] but hard to describe in words. Imagine that the random switching of cell states is simulated by spattering P-, V-, or D-colored “paint” onto a tricolored pie chart. Wherever the “P + V + D = T” criterion is satisﬁed, a tip will grow out. Among the possible conﬁgurations are 1. A dab lands wholly inside a sector. In this case, nothing happens because there would only be a bipartite reaction (P + V, V + D, or D + P) at the spot’s edge. 2. A dab lands at the perimeter and straddles a boundary (Fig. 5.3g). If the spot’s color matches a ﬂanking color, then nothing happens, but if it differs from both, then T will be made where the three colors meet, and an extra tip will emerge in mirror image to the endogenous tip (for reasons described in 3). 3. A dab straddles a boundary but does not reach the perimeter (Fig. 5.3h). If the spot’s color differs from both ﬂanking colors, then T will be made wherever its edge crosses the border. Rounded spots will have two T points, and the line connecting them must intersect the disc’s center because they lie on a radius (P/V, V/D, or D/P). Thus, the ﬁrst part of Bateson’s Rule is satisﬁed: the three tips will lie in one plane. Because each T point has a pinwheel (PVD) handedness, a person in the P zone near a juncture must walk either clockwise (L) or counterclockwise (R) to step into V and then into D. Along a radius crossed by a dab, the handedness changes at every T point, thus fulﬁlling the second part of Bateson’s Rule (i.e., R-L-R or L-R-L alternation). 4. A dab’s edge touches, but does not cross, a boundary (Fig. 5.3i). T will be produced on either side of the intersection, and a pair of tips begins to grow out but eventually converges (vs. diverges as in 2) because (1) “the distance between the two [tips decides] . . . how many elements are missing . . . due to overlap of the two gradients,” or (2) “the normal maximum concentration is not achieved [because] . . . too few

101

cells of a particular compartmental speciﬁcation are available or if they are not close enough” [2804]. The leg’s bristle pattern is too rich to be encoded by three broad angular values (P, V, D sectors) [3253]. Meinhardt recognized this problem and in a 1983 paper sketched a “Hybrid PC-Boundary Model” (Fig. 5.3k) where the P/V, V/D, and D/P lines provide three angular values in the nascent disc [2808], with remaining values emerging later via the Shortest Intercalation Rule. One difﬁculty with this remedy is that all three spokes lie outside the coordinate-dense UM quarter, so it should be unable to regenerate. In the same article, Meinhardt concocted an alternative solution, which will here be called “Boundary Model II” (Fig. 5.4). Its key amendment was that “the compartment borders must act as a frame for the ﬁner circumferential subdivision, [with the] distance from a particular compartment border [being] measured by a diffusible morphogen” – an idea earlier suggested by Francis Crick and Peter Lawrence [904]. Echelon 1 (at the boundaries) P+V=A V+D=B D+P=C Echelon 2 (at the center of the disc) A+B+C=T T would thus be created in two steps (vs. one in Boundary Model I), and its source point would be deﬁned by the intersection of three lines (vs. the juncture of three areas). More important, an angular coordinate could be cobbled from the new factors. A, B, and C are supposed to be long-range morphogens (like T but unlike inducers P, V, and D), so they could form tentshaped gradients [2804]. Cells could then ﬁgure out where they are by measuring concentrations of A, B, and C. To encode angular PI, each gradient must curve around the circumference (Fig. 5.4a), a constraint that a priori seems implausible [618, 2456]. Conceivably, the molecules could be channeled or actively transported (see below). Cartesian and polar systems could easily be interconverted [922, 1298, 2157, 3252, 3703], but their coordinates cannot be swapped directly inter se [548, 2512, 2881, 3915, 4697]. Boundary Model II also explains why some disc fragments duplicate (Fig. 5.4b). Thus, for example, if the D sector is excised, then the P and V edges should synthesize A (when they heal together) at a site opposite to the extant A source [2808]. If the new A gradient respeciﬁes cells by morphallaxis, then it will cause a mirror-image

102

IMAGINAL DISCS

a 8 910

11 12/0

b678

1

7 6 5

e dcb

a

1

4

L

R

L

L

1

1

L

2 4

1

b

7 6 5

L

2 4

3

2

L

3

k D V

R L

P

P

P

explains:

D

88%

... but not:

D

D

D

P

V

P

25% V

V

0%V

P

D

D

16% P

D P

19% V

L

V

D

L R

V

D

D

V P

a e dcb 2 P 3

hybrid

h i

j

V 4

P

V

D

6 5

g

conical gradient

L R L

9 10 11 12/01

7

P V

10 1112/0 11 10 11 9 11 L 12/0 8 12/0 1 12/0 a 7 bR 1 1 6 1 2 5 2

8

Boundary Model D

3

4

3

Polar Coordinate Model

f

2

4

3

d e8 910 1112/0a

10 11 12/0 9 8 7 6 a 5 b

1

L

2

4

3

R

1

2

3

10 11 12/0 9 8 7 7 6 b5 9 8 8 10 4 7 11 3 12/0 6 5 1 a 2 4 5 4 3

11 12/0

7 6 5

5

2 4

c 8 910

w

12/0 2 1 11 3 11 12/0 4 10 9 5 6 8 7 6 5

ne

10 11 12/0 12/0 9

V 80% P

Paradox

CHAPTER FIVE. THE LEG DISC

duplication. In accord with this scenario (but not with the epimorphosis required by the PC Model), duplicating fragments grow negligibly and lose structures near wound edges [20].

The Boundary and PC Models jousted in a ‘‘Paradigm War’’ Given the reasoning in the original Boundary Model, distal outgrowths should only sprout from dorsal, ventral, or anteroventral faces of the leg (P/D, P/V, and D/V radii). In this regard, it surpassed the PC Model (where constraints are looser) at explaining why outgrowths tend to lie in the D-V plane [617, 4490]. It also afforded a simpler view of how a disc’s coordinate system might be “geared” to the segmentation gene machine of the embryo [2812, 2813]. To wit, the A/P boundary in each thoracic segment would be recruited as a reference axis for each leg or wing disc [2808] – a conjecture veriﬁed by

103

later molecular studies (Fig. 4.4) [834]. Ironically, Boundary Model II was stumped by the same Quadrant Regeneration Mystery that the PC Model handled relatively easily (Fig. 5.3j) [2804]: how could D-type cells alone be able to regenerate a tip? Despite their ability to “cross-hybridize” (Fig. 5.3k) [1807, 2814, 3253], no two models ever differed so fundamentally as the Boundary and PC Models [834]. Their rivalry centered on several pivotal issues [354, 1807]. 1. Who’s talking and who’s listening? According to the PC Model, every cell identiﬁes itself to its neighbors and receives such input in return. In the Boundary Model, only a few cells emit signals; most are silent receivers. 2. Do cells shout or whisper? For the PC system to work, cells need only converse with their neighbors, and they are thought to do so by direct contact [1301],

FIGURE 5.3. Models for pattern formation that were prominent in the 1980s, and how they explained regeneration-duplication

phenomena in leg discs (cf. Fig. 5.2). In the Polar Coordinate (PC) Model (a–e) [1303], growth is supposedly stimulated when distant cells abut via healing, whereas in the Boundary Model (f–j) [2804, 2808] distal growth is provoked when three kinds of cells convene. a. Polar coordinates in a left leg disc (cf. Fig. 4.6). Each cell assesses its position as (1) radial distance (lettered rings) from the center and (2) angular declination (numbered spokes) from an arbitrary radius (12/0). Unlike the wing disc where angular values are presumed to be spaced evenly, here they must be crowded because the UM quadrant regenerates. b. In the PC Model, fragments (e.g., UM) with >50% of the angular values regenerate (black triangles), whereas pieces (e.g., 3/4 part) with <50% duplicate (hollow triangles). In each case, the edges (5 and 12/0) intercalate via the shortest route (cf. Fig. 4.6d). c–e. Distal outgrowths can occur if tissue is lost from certain regions, and the edges of the hole (white “sausages”) heal as shown. c. If cells are lost from the UM perimeter, then an extra “clockface” arises when new tissue (black) ﬁlls the 5–12/0 gap by the shorter route (viz. 1–4). Because the new clockface is backward, the outgrowth will be right handed (R vs. L). Comparable losses from other peripheral parts should not form whole circles, which is problematic because mirror planes of twinned legs tend to be at right angles to spoke 4, not 8 [3442]. d. Loss of internal coordinates can generate two whole new circles if wound edges reorient (during evagination?) to abut values from ≥50% declination (e.g., 9 and 3; “a” and “b” = outer two rings) [1495]. e. Confrontations of <50% declination will form partial circles and stunted outgrowths. L-R-L handedness (d, e) obeys Bateson’s Rule (see text), but the model does not explain why converging outgrowths (e) occur near angular value 1 (cf. Fig. 5.2c) and not near 2–4. f. In Meinhardt’s Boundary Model, the anterior compartment is subdivided into ventral (V) and dorsal (D) parts, whereas the posterior (P) compartment is not subdivided. The PVD sequence is clockwise in left legs (curved arrow). Each cell identity – P, V, or D – furnishes one key ingredient for making a morphogen. The morphogen is created only at the nexus, and its diffusion forms a conical gradient (rings = contours; epithelium in side view). g–i. The model explains outgrowths. g. Duplications arise when a peripheral patch that straddles a boundary (e.g., D/V) transforms into the third type (P), thus creating a new PVD nexus (arrow) that grows out to form a right limb. h. If such a patch is internal, then limbs arise at two new PVD points. i. If the patch merely touches a border, then too little morphogen may be made to sustain two tips, in which case the outgrowth converges. As for why ventral outgrowths diverge and dorsal ones converge (cf. Fig. 5.2c), the model must assume (ad hoc) that D spots tend to straddle P/V (h), whereas V spots only touch D/P (i). j. The model (in a leg disc at left) explains most surgical outcomes. When the boxed fragments are cultured, they regenerate claws (bicurved symbol) and other distal structures that map in the excised endknob at frequencies indicated. Regenerative ability is maximal when all 3 domains remain (88%), as expected. However, the model does not explain why the UM quadrant (sans endknob) also replaces claws often [2804]. k. Hybrid PC-Boundary Model, where P/V, V/D, and D/P radii specify angular values (4, 5, 1) in a PC system [2808]. Panels a and b are redrawn from [1303] (angular spacing was later revised [4140]); c–e are adapted from [1495, 1496, 3442]; f–k are based on [2804, 2808] (he uses AD and AV, not D and V); data in j are from [3811, 4140]. See also App. 7.

IMAGINAL DISCS

D

gradie ved nt r u C

b

s

a

P+V=A V+D=B D+P=C

c

104

A+B+C=T

B V

A' V

A conical gradient

Boundary Model II

c dAC

b

gradie ved nt ur

Hh + vAC = Wg Hh + dAC = Dpp

L

Dpp

Wg + Dpp = T T

PC Hh

P

A

(T>5) = Dll

Hh

vAC

V

P

A

s

c

P

T

P

Hh-Dpp-Wg Model

V A

Dll

Wg Arc Scenario

?

n

ra

Cloud Scenario om diffusio nd Dpp

?

Wg FIGURE 5.4. Molecular validation of the basic premises in Meinhardt’s Boundary Model.

a. The Boundary Model II (as revised by Meinhardt in 1983 [2808]). In this version (cf. Fig. 5.3), there are 3 new morphogens (A, B, C). Each of them arises at an interface (P/V, V/D, D/P) where region-speciﬁc molecules (P, V, D) interact after diffusing a short distance into the adjacent region. A, B, and C then react at the center to form the tip morphogen T. Finally, T diffuses to form a conical gradient that provides the radial coordinate of a polar system, while the angular coordinate is speciﬁed by the graded concentrations of A, B, and C. The gradients must overlap (not shown) to encode every angle uniquely. b. According to this model, a VP fragment should duplicate because V and P will create a second source of A (A ) when they heal together. c. Hh-Dpp-Wg Model. Developing legs actually use a strategy like Meinhardt’s model, although the ﬁrst induction involves one-way (not bidirectional) diffusion (arrowheads), and there are only 2 (not 3) long-range morphogens. Cells in the Posterior Compartment (PC) make Hedgehog (Hh) – a short-range signal. When Hh diffuses across the A/P border, cells in the ventral AC (vAC) respond by making Wingless (Wg), while dorsal (dAC) cells make Decapentaplegic (Dpp). How Wg and Dpp travel is not known. One idea (Arc Scenario, above) is that they move in arcs to form curved gradients, with subsequent steps (e.g., speciﬁcation of angular values) as in Boundary Model II [1807]. The gene Distal-less (Dll) is shown being activated above a threshold (5) in the T gradient (assumed to be 10-to-1 centrifugally). Another idea (Cloud Scenario, below) is that Wg and Dpp diffuse randomly [618, 2456], in which case they could activate downstream genes directly (rightmost arrow) where their clouds overlap (via the rule “Wg + Dpp = Dll”) without any intermediate tip morphogen [2813]. Indeed, one Wg target gene (Dfz3) does appear to be expressed in a parabola-shaped domain [3977]. However, defect arcs in genetic mosaics [1811] are more easily explained by the former scheme (see text). The Arc Scenario may also apply to the developing notum (cf. Fig. 6.14), where the dpp-on stripe is at a ∼30◦ angle relative to where its target gene (wg) is turned on [4369]. Panels a and b are based on [2808] (p. 381, para. 2), and c is adapted from [618, 1807, 1991, 2456].

CHAPTER FIVE. THE LEG DISC

whereas in Meinhardt’s scheme, cells must broadcast signals over distances of tens or hundreds of cells using diffusible molecules as messengers. 3. Is the coordinate system seamless? This issue has been debated ever since compartments were discovered in 1973 [2161, 2513]. In the PC Model, the circumference has no discontinuities. In Boundary Model II, compartment boundaries are distinctive as reference axes (as in a Cartesian system) [2808]. As the Cellular Epoch drew to a close in the late 1980s, evidence was mounting for the Boundary Model. Peculiarities of the A/P border (e.g., the two below) were difﬁcult to reconcile with the PC Model’s notion that all angular values are equivalent. The leg disc offered the ideal battleﬁeld because its geometry ﬁts a polar coordinate system so neatly (leg segments = rings, and bristle rows = spokes; Fig. 5.1c) [1311, 1807], and its A/P line is as inviolate as the wing’s [4076, 4254]. Nevertheless, the wing disc was better known in terms of growth and lineage, so some of the skirmishes took place there instead. Certain properties of the A/P border were problematic for the PC Model. For example, regenerative ability was found to be correlated with the cut’s location relative to the A/P line. Compartments had originally seemed irrelevant for PI because they can be reestablished during growth of wing- and leg-disc fragments [5, 354, 2213, 4223] or during apoptosis-induced duplication of legs in situ [1504, 3701], and Bryant’s map of regenerative potency revealed no discontinuity at the A/P line (Fig. 4.5). However, when Jane Karlsson reinvestigated using other cuts, she found a clustering of angular values near the wing’s A/P boundary [2140]. No such correlation is seen in the Drosophila leg disc [4140], but circumferential intercalation in the legs of other (hemimetabolous) insects is constrained by A/P lineage boundaries [1297, 1299, 1301, 1302]. The PC Model was also unable to explain why the A/P border region is a haven for slower growing cells in the wing blade. In the developing wing (although perhaps not elsewhere in that disc [1431, 2935, 4703]), cells that divide more slowly than their neighbors are winnowed from the population [2229, 2935, 3949, 3961, 3962], except near the A/P and D/V compartment borders [3947, 3948]. “It is as if cells at a compartment boundary form a competitive pool that is separate from that of the internal cells” [1639]. In the case of the leg disc, the gynandromorph fate map is compressed on either side of the A/P boundary [1800] – indicating less mixing of unrelated cells

105

there – but this trend might not involve competition per se [3961]. At the dawn of the Molecular Epoch, many genes were found to be expressed in sectors (Fig. 5.8) or rings (Fig. 5.11) within the leg disc. These patterns were hailed as conﬁrmation of the PC model [531, 880, 1304, 1574, 1970], but subsequent analyses of the gene circuitry tipped the scale in favor of Meinhardt’s model [617, 2814]. The awkward truth, however, is that much of the new data does not ﬁt neatly into either scheme. Chief among these “ugly facts” are the mirror-image duplications manifest by wg LOF and dpp LOF legs (Fig. 5.5), which imply a bipolar coordinate system [1812].

Hh, Dpp, and Wg are the chief intercellular signaling molecules Hh, Dpp, and Wg are all secreted signaling proteins. Hh and Wg are founding members of the Hedgehog [1701] and “Wnt” (Wingless and Int-1 [3148]) families [3923], while Dpp is a member of the TGF-β family [3099]. During the 1990s the molecular players in each transduction chain were ﬁtted together, like clues in a Sherlock Holmes mystery, to reveal the routes from the ligands to the nucleus. Among the surprises in these pathways were 1995

1996

1997

1998

1999

Discovery (in several labs) that Hh transduction involves a cAMP-dependent kinase (DC0 encodes its catalytic subunit) whose only known developmental role until then was in slime molds [355, 2115, 3343]. Identiﬁcation of the long-sought Wg receptor (Dfz2) [310, 1971, 3656] and Hh co-receptor (Smo) [61, 4441], which were unexpectedly alike [3203, 3344, 4708]. Identiﬁcation of Pan as the ﬁnal link in the Wg chain [519, 1088, 4439], which solved one mystery but led to a deeper one: why should the pan (Wg pathway) and ci (Hh pathway) genes be adjacent (∼10 kb apart)? Realization that the Hh and Wg pathways also share an unsuspected Slimb-dependent proteolysis of their transcription factors (Ci and Arm) [2060, 4279]. Revelation that the proteoglycan Dally is a co-receptor for both Wg [641, 2556, 3309, 4400] and Dpp [2004, 4400] – hence challenging the idea that Wg or Dpp are freely diffusible, as had earlier been disputed for Hh [277, 4275].

Figure 5.6 sketches the Hh, Dpp, and Wg transduction pathways. The players within each pathway are

106

IMAGINAL DISCS

a

b PAB

c wg

LOF

d dpp

LOF

D

6 7 8

5

W g1

2

V

V

Dpp 4 3 2

7 6

6

5

5

4

8

4 3

3 2

D/D

1

7

2

8 1

V/V

6

5

4

7

D

V/-

5

8

3 1

2

dpp

Cl

LOF

Deficiency & duplication Deficiency only FIGURE 5.5. Phenotypes of wg LOF and dpp LOF legs that argue for a bipolar coordinate system instead of Meinhardt’s 3-azimuth model. For gene abbreviations, see App. 6. a. Anterior face of a wild-type left 1st leg from midtibia to the distal tip. Ventral (V) and dorsal (D) sides face left and right. Landmarks include pre-apical bristle (PAB), sex comb, and claws. The apical bristle (not labeled) is less prominent on 1st (vs. 2nd) legs. At right is a fate map (cf. Fig. 5.1c). Inner circle is the tarsus (1–8 are bristle rows). The sex comb begins as a ventral transverse row; claws and PAB are dorsal [1812]. Crooked white line delimits Dpp (∼225◦ sector) and Wg (∼135◦ ) “domains of inﬂuence” (bounded by rows 2 and 7; cf. Fig. 5.2c), based on LOF defects (b–d). Because these sectors are so much larger than the slivers where dpp and wg are transcribed (cf. Fig. 5.4c), Dpp and Wg must act as secreted signals [834], but whether they are bona ﬁde morphogens is debatable. These sectors may actually be more equal in size because D bristle rows are closer to one another than are V rows (cf. Fig. 5.1e). b. In wg LOF legs, V structures are missing and replaced (cf. key at bottom) by a set of D structures arranged in mirror symmetry [880, 1812]. This D/D (“Janus”) symmetry extends through the sternopleura [177]. D/D legs also arise in arm LOF [3317], dsh LOF [2262], gam LOF [599], pan LOF [519], porc LOF [2110], and sgl LOF [1673] ﬂies and are inducible by wg null [1811], arm null [3317], and probably arr null [4570] V clones (cf. Fig. 5.6). D/D legs can also be created by heat-pulsing t.s. NLOF larvae [886] – a perplexing result because there is no known role for the Notch pathway in Wg signaling in legs. The few sex comb bristles that remain fail to rotate. A milder D/D duplication is seen in Egfr LOF gro LOF double mutants [3465]. Finally, various kinds of treatments that cause trauma (microcautery, irradiation, etc.) can produce D/D phenotypes [425, 2102, 3442, 3449, 3705], presumably because the dpp-on D sector has more robust growth potency than the wg-on V sector (cf. Dorsal Remnant Mystery, Fig. 5.2b) [1807]. c, d. In dpp LOF legs, D structures tend to be missing from the tibia and tarsus [1812]. Often they are replaced with V structures (c), but sometimes not (d). c. V/V phenotype. When a second set of V structures arises, the ﬁrst legs have U-shaped sex combs. Janus V/V legs are also seen in punt LOF [3932, 4277] and tkv LOF ﬂies [753] and are inducible by dpp null [1811] or punt LOF D clones [3329]. d. “V/-” phenotype. Missing structures in dpp LOF legs are commonly not replaced. (No analogous “D/-” phenotypes are seen with wg LOF legs.) In such cases, the V remnant tends to shorten and curl dorsally. These ﬁgures are based on [177, 1811, 1812, 3317].

CHAPTER FIVE. THE LEG DISC

outlined in App. 6. Some familiarity with these molecular components is essential to grasp the tissue-level narrative that follows.

P-type cells use Hh to ‘‘talk’’ to A-type cells nearby Based on detailed analyses of the above pathways, we now know the raison d’ˆetre for A and P compartments in most discs: they establish separate groups of “signaler” and “receiver” cells [354, 2448, 4488, 4848]. The P group emits a signal (Hh) to which the A group can respond [571, 1078, 4229]. Limited diffusion of the Hh signal ensures that only a subset of A cells (those at the border) will actually respond [358]. This “Short-range Inducer Model” thus relies on a principle that henceforth will be called the “Deaf-Speakers/Mute-Listeners Trick.” A vs. P identities in thoracic discs are implemented by two different transcription factors: En (homeodomain class) [1031, 3719, 3860] and Ci (zinc ﬁnger class) [65, 155, 1827, 3194, 4503]. In P cells, En somehow activates hh transcription [1647, 1659, 4227, 4848] so P cells secrete Hh. Simultaneously, En represses ci transcription [1135, 1647, 2581, 3818] so P cells are refractory to their own signal (because Ci is needed for Hh transduction). All A cells can detect the signal because they express every transduction component (Ptc, Ci, etc.), but most A cells should be unable to emit Hh because Ci-75 represses hh [155, 1078, 2832]. The asymmetric roles of A vs. P cells in this discourse [354, 2448, 4848] contrast with the mutual speaker–listener roles envisioned by Boundary Model II [2808], but the two models are similar beyond this point [1427, 3497]. When secreted Hh spreads across the A/P line [1472, 4228], it rescues the activator form of Ci (Ci-155) from the proteasome guillotine so Ci-155 can convey its signal to target genes. In summary, three distinct zones are maintained along the A-P axis of each thoracic disc as a result of this circuitry (Fig. 5.7): 1. P-type cells express En, which activates hh but suppresses ci and dpp [4229]. En’s inhibition of dpp occurs by two routes: En directly binds dpp’s cis-enhancers [2980, 3747], and En blocks ci expression so Ci-155 cannot activate dpp [1647, 1827, 2832]. 2. A-type cells at the border receive Hh signal that diffuses from the P side. They respond (via PKA and other agents) by rescuing Ci-155 from being cleaved so it can activate dpp and ptc. 3. A-type cells beyond the range of Hh express ptc at a basal level [2115], and Ptc (by a double-negative route) relieves repression of PKA. PKA then phosphorylates Ci to provoke proteolysis of Ci’s transcription-

107

activating (dCBP-binding) domain, and the remnant (Ci-75) keeps hh, dpp, and other target genes off. The en-off state of A cells is inherited from the embryo [1093, 1124, 1790, 3129] (cf. Fig. 4.4). It is maintained by Polycomb [582, 2889, 4327], polyhomeotic [2728, 3512, 3753], dRaf [2532], fat-head [4607], and groucho (D/V boundary only) [998], so no further control by Ci-75 is needed. Hence, the Ci-vs.-En circuit is asymmetric: En keeps ci off in P cells, but Ci-75 does not keep en off in A cells (ci null clones in the A region do not turn en on [1078, 2832]). The en-on state of P cells is maintained by trithorax [452], moira [475], polyhomeotic [2728], and the EGFR pathway [207], rather than by the autoregulatory “En en” loop seen in embryos [1790]. Indeed, En en in discs [207, 1647, 4229], although this GOF effect may be spurious relative to the circuit’s normal range of operation [156]. It is possible to artiﬁcially drive ci expression (via act5c-Gal4:UAS-ci) in en-on (P-type) cells so as to make them co-express both Ci and En [943]. Such schizophrenic cells “hear themselves talk” (via “Ci-155 Ptc” and via “En Hh”) and behave like border cells [943]. In contrast, deaf-mute (en null ci null ) cells behave uniquely (vs. A or P or border cells) in terms of adhesive afﬁnity (cf. Ch. 6) [943]. Other questions pertain to Ci-75 vs. Ci-155, which have identical DNA-binding domains [2980]: 1. Because hh is a target gene of Ci-75 [2832], Ci-155 should turn hh on at the border [155], but it does not. Evidently, hh is more sensitive to inhibition by Ci-75 than to activation by Ci-155 (both of which exist there) [2832], but how this selectivity is achieved physically is unclear [2980]. Oddities of this zone suggest that other factors might override these controls [2728, 4478, 4539]. For instance, ectopic Ci-75 can repress ptc there but not dpp [155]. 2. A minimal amount of ptc transcription in A cells is needed to keep dpp off beyond the border zone [642], but if this baseline were due to a constitutive promoter, then ptc should also be expressed in the P region. So why isn’t it? Apparently, ptc’s expression in A cells stems not from its promoter per se but from an unknown A-type activator aside from Ci-155. Because anterior ptc expression does not increase when ci is removed [2832], ptc must be selectively “deaf” to Ci-75 (just as hh is deaf to Ci-155). Again, the basis for such selectivity is obscure [2980].

heteromeric receptor

Tkv

ks?

P

? enz microtubule

?

P

? P

Fu

cog

?

PKA

Cos2

primes for cleavage

~kinesin P

tr. factor Ci

cyt.

Slimb

P

cyt. . nuc

APC

dCBP co-activator

Arr

Dsh enz

enz

Sgg

PP2A P

cog cog

repressor

dCBP Brk

activates

P

dAxin

cog

Shn

repressor activates Target genes

n

P

Medea

co-activator

uc.

?

enz

? PKC

Mad tr. factor

Dfz2 cog

P

P

co-activator?

?

? CK2

blocker

?

PP2A

Su(fu)

?

receptor

Dad

P

enz

?

enz

mas

?

ligand Wg

Slimb

junction

co-receptor

Punt

?

Smo

Ptc

receptor

?

c Wingless pathway

primes for proteolysis

P

Shg

diverts into junctions

Arm co-activator

Target genes

Key binds activates inhibits phosphorylates de-phosphorylates

P

P

Dally

ligand Dpp

ligand Hh

Dally

b Dpp pathway

co-receptor?

a Hedgehog pathway

co-receptor?

108

Pan tr. factor

Gro co-repressor

activates

Target genes

CHAPTER FIVE. THE LEG DISC

Hh elicits expression of Dpp and Wg along the A/P boundary In 1994, the Short-range Inducer Model was invoked to explain Hh’s effects in leg discs. Hh was shown to turn on wg in the ventral half of the border, and to turn on dpp in both the dorsal and ventral halves (Fig. 5.8) [231, 1037, 2466]. Ventral dpp transcription, which is relatively meager [2739], turned out to be a red herring from the standpoint of patterning since dpp null V clones, unlike D ones, are anatomically normal [1811]. For some unknown reason, the dpp [2739, 2954] and wg [177, 3317] transcription zones are shaped like sectors, rather than bands. The dpp-on domain is narrower than the wg-on wedge and hence is often called a “stripe” [487, 3747]. The width disparity could trivially be due to dpp turning on at a higher Hh threshold, but it might exist for other reasons (e.g., dpp-on cells adhering more tightly or dividing more slowly). The sectors coincide roughly with the leg’s prospective D (dpp) and V (wg) midlines. If, as argued below, Dpp and Wg are morphogens for specifying positions along the D-V axis, then this state of affairs would explain the Gaps Mystery. To wit, the reference line would not be the A/P compartment boundary per se as ﬁrst thought [904], but rather the slightly offset line that bisects the dpp and wg transcription sectors (Fig. 5.4c). Embryos also manifest Hh-dependent Wg expression (cf. Ch. 4) [3386], but Dpp is not under Hh control at that time. In comparing a Wg blastoderm stripe to

109

the Wg sector of the leg disc (and doing the same for En [3747]), the leg disc seems equivalent to a body segment (or part thereof) that has curled itself into a circle [880, 1039]. This basic topology agrees with both the PC and Boundary Models, as well as with the Angular Values Hypothesis [2291]. Viewing the nascent disc as a circularized segment is appealing because it provides a simple way to co-opt the established intercellular signaling machinery of the segment polarity genes for circumferential patterning in the discs. [834]

The leg disc also resembles a body segment insofar as Dpp apparently acts as a dorsalizing agent in both systems (see below) [1807]: in the embryo, a Dpp gradient speciﬁes diverse fates within the dorsal 40% of the ectoderm [127, 1091, 1211, 4613]. Why should Hh turn on different genes in different parts of the A compartment? The answer is that dorsal (dAC) and ventral (vAC) cells of the A compartment differ in competence (Fig. 5.4c). These biases are revealed when Hh is expressed ubiquitously: Hh then activates dpp throughout dAC and wg throughout vAC [231, 617, 1037, 2466], rather than just along the A/P boundary (Fig. 5.9c). Activation of dpp in dAC (and weakly in vAC) and wg in vAC is also seen in somatic clones where the Hh pathway is turned on downstream of Hh by LOF mutations in patched [2058, 2491] or DC0 [2058, 2059, 2491, 2533, 3238]. For slimb LOF clones, the situation is more complex because slimb also functions in the Wg pathway [2060, 2856, 4279].

FIGURE 5.6. Some key signaling pathways in disc development. In the working models depicted here, certain components may

be replaced by others (not shown) in different tissues, regions, or time periods. Three cells are depicted, with apical microvilli at top, although receptor-ligand interactions may actually take place elsewhere on the surface. Black rectangles = proteins; gray area = cytoplasm (“cyt.”); dashed line = nuclear membrane. Arrows (activation) and blunt “ ” lines (repression) indicate epistatic relations. Noncovalent binding is symbolized by interlocking hooks (cf. key). Question marks concern how or whether the interactions occur (see text). A “cog” (a.k.a. “adaptor”) is a component that plays a steric (nonenzymatic) role [441, 1954, 3299, 3676] (e.g., Slimb [2668]). Enz = enzyme, CK2 = Casein Kinase 2, PKA = Protein Kinase A (likewise PKC), Shg = Shotgun (a.k.a. dE-cadherin). For other abbreviations, see App. 6. For perspective, see App. 7. a. Hedgehog pathway [1554, 1974, 2075, 3685, 3831]. Binding partners (listed from surface to DNA) are based on ﬂy studies or vertebrate homologs (vh): Hh-Ptc [755] , Ptc-Smo (vh [4130]), Fu-Cos2 [4068] , Fu-Ci [4068], Fu-Su(fu) [4068] , Cos2-microtubules [3612, 3976], Cos2-Ci [4068, 4536] , Su(fu)-Ci [4068] , dCBP-Ci [54], Ci-DNA [751] . Ci’s interaction with dCBP may be inhibited by PKA (link not shown) [54]. b. Dpp pathway [959, 1983, 3498]. Dally is placed upstream of Dpp, based on the ability of extra doses of dpp+ to suppress dally LOF phenotypes [2004], and a similar argument applies to Wg [2556] . Evidence for binding: Dpp-Punt [2495], Dpp-Tkv [2495] , Dpp-proteoglycans (e.g., Dally) [2004] , Punt-Tkv [2495], Tkv-Mad [1983], Tkv-Dad [1983], Tkv-PP2A (vh [1627]), Mad-Medea [1983, 4703], Mad-dCBP [4534], Mad-DNA [2217]. Mad may also bind microtubules (not shown) [1084]. The “Shn brk” link does not actually occur at the protein level, but rather occurs via inhibition of brk transcription [2727]. c. Wingless pathway [422, 3919]. Arr is thought to be a co-receptor for Dfz2, but it does not “present” Wg in the same way as Dally [4570]. Conceivably (not shown), Arr may act via CK2: Dfz2 Arr CK2 Dsh, etc. The APC homolog here (labeled simply APC) is likely E-APC [4832]. Sgg is thought to be Arm’s natural kinase [3683] (but see [3228, 4062]). Evidence for binding (see [3316] for details): Wg-Dfz2 [310], Wg-proteoglycans like Dally [598] , CK2-Dsh [3149, 4676], Dsh-dAxin (vh [2238] ), Sgg-APC (vh [3675]), Sgg-dAxin [3683], APC-dAxin [1698], PP2A-dAxin (vh [1919]), dAxin-Slimb (vh [2241]), APC-Arm [4832], dAxin-Arm [1698], APC-Arm [4832], Slimb-Arm (vh [4701] ), Shg-Arm [3749, 4268, 4416] , Arm-Pan [519, 4439], Gro-Pan [692, 2496], Pan-DNA [519, 4439].

110

IMAGINAL DISCS

Leg disc

a

Wing disc

D A

P V

dorsal region

b

A P En

c border zone

Smo

Ptc

Smo

PKA

ptc

PKA

Hh

hh

diffuses

ptc

(low)

P

Ptc

en

En

P P P P P

Ci-155 e ot pr

Ci-75

om as

Zn

dpp

Zn

dCBP

e

dpp dpp

d en ci dpp

hh omb

diffuses

Dpp

diffuses

omb

ci

CHAPTER FIVE. THE LEG DISC

The dAC/vAC border is not a strict lineage boundary [487, 1800, 4076], so dAC-type cells can presumably jostle into vAC territory and vice versa. No genes have been identiﬁed that are expressed strictly within dAC or vAC [3919], nor are any genes transcribed along the dAC/vAC border (as expected based on Boundary Model II) [4277], so the nature of these areas is not obvious [617].

Dpp dorsalizes and Wg ventralizes, or do they? Conceivably, dAC and vAC states are maintained by the Dpp and Wg signals themselves [487, 2954, 4277]. That is, AC cells within range of the Dpp signal adopt dAC competence, while those within range of Wg adopt vAC competence. Such a system could be stable without celllineage restrictions (cf. the Cabaret Metaphor). Support for this idea comes from an experiment where cells are tricked into switching states: when dAC cells are duped

111

(via sgg LOF ) into thinking they are receiving a maximal Wg signal, they stop making Dpp and activate their own wg gene whenever they are within range of Hh [1833, 2059] (notwithstanding a report to the contrary [1039, 4487]). Additional evidence is provided by the fact that the dAC/vAC border (which runs along row 7 in the fate map; cf. Figs. 5.1c and 5.4c) coincides with the line that separates the Dpp and Wg “spheres of inﬂuence” (Fig. 5.5a): in dpp LOF mutants the tarsus lacks structures on the D side of “L2&7 ” (the line that runs along rows 2 and 7), whereas, in wg LOF mutants the whole leg loses structures on the V side of L2&7 (Fig. 5.5b-d) [1812, 3317]. L2&7 is also deﬁned by leg defects caused by dpp null or wg null clones [1811]. It divides the disc into two sectors that would measure ∼225◦ (D) and ∼135◦ (V) if bristle rows were evenly spaced around the leg (cf. a femoral septum at this angle [3867]), but in fact there is a larger space

FIGURE 5.7. Effects of Hh diffusion into the A compartment of thoracic discs. For gene abbreviations, see text.

a. Left leg and wing discs, with D-V axes of the central regions oriented vertically (cf. compass). A and P compartments are lightly or darkly shaded, respectively. The boxed areas of both discs obey the “Hh Dpp” link, but the ventral part of the leg disc obeys “Hh Wg” instead (not shown). b. Part of the disc epithelium that straddles the A/P compartment boundary (magniﬁed from a). Hexagons are cells (although packing is not really so orderly). Cells in the P region express the transcription factor En, while A cells do not. Zigzag line marks the anterior limit of the dpp-on zone. c. Circuitry of genes and proteins (abridged; cf. Fig. 5.6) that operates in each of the cell types delineated in b. The chain of events (right to left) starts with expression of en in all P cells. (P cells have Smo but not Ci or Ptc [1022, 2832].) En activates hh but represses ci and dpp [1647, 2832, 2980, 3747, 4227]. Hh diffuses ∼8–10 cell diameters into the A region [4136]. Binding of its receptor Patched (Ptc) triggers synthesis and release of Dpp, which turns on genes like omb (cf. Fig. 6.3) [2455, 3074]. The “Hh Dpp” link is mediated by the transcription factor Ci (horizontal bar). Full-length Ci (155 kD, 1396 a.a.) has 5 tandem zinc ﬁngers (Zn), a C-terminal domain that binds the co-activator dCBP, a proteasome cleavage site (zigzag line), 5 residues capable of phosphorylation by Protein Kinase A (vertical lines) and other domains (cf. text) [54, 2832]. A-type cells do not express dpp in the absence of Hh (left) because basal transcription of ptc [4136] supplies enough Ptc to derepress PKA (via Smo). Phosphorylation of Ci by PKA leads to Ci’s proteasome-dependent cleavage, and the N-terminal fragment Ci-75 acts as a repressor (due to an alanine-rich section [65]) of dpp and hh [2832]. Dpp and Hh are both diffusible (triangles denote gradients), but Dpp’s range is greater (cf. Fig. 6.3). Omitted here is the enigmatic transcriptional regulation of ci by the zinc-ﬁnger protein Combgap (Cg) [621, 4017, 4217]: Cg ci throughout the A region of wing and leg discs, but Cg ci in the P region of wing (but not leg) discs. Curiously, the En hh link is inoperative in the developing eye [4168]. d. Summary of on/off states (thick vs. thin lines) of en, ci, and dpp. The dpp-on stripe is evidently “painted” in two ways: (1) directly via “Hh dpp” and (2) indirectly by a ﬁgure-ground “illusion” [657, 1585, 3747], where “Ci-75 dpp” anteriorly and “En dpp” posteriorly. Both actions stem from the disc’s en-on/off duality. Validation of this logic comes from larvae carrying an en transgene driven by a heat-shock promoter. When such larvae are heat-shocked, every cell transiently becomes en-on (P-type) and the dpp-on stripe disappears [3747]. A similar shut-off is expected for discs devoid of En, but this condition has not yet been created [1839]. Not shown is a trespass of the en-on edge into the border zone that occurs in late 3rd instar when a “Hh en” link pervades the disc [1647]. (Its ubiquity is shown by the ability of ptc null A clones turn on en [4136].) N.B.: Events in the boundary zone are more complex than depicted here (cf. Figs. 6.3–6.5), especially for Ci [4540]. En’s inhibition of dpp (En dpp) occurs both directly (by binding of En to dpp’s cis-enhancers [2980, 3747]) and indirectly via En’s suppression of ci (because Ci-155 dpp [1827, 2832]). Suppression of ci is also partly mediated by Polyhomeotic [3512] – a member of the Polycomb Group of repressors (cf. Ch. 8) [1012, 1867, 2374]. En’s activation of hh (En hh) is probably indirect (En X? hh) [224] because En normally acts a repressor [2047, 2048, 3175, 3860, 4003] by recruiting Groucho [1243, 2064, 4351] (but see [1706, 2642, 2729, 3481]). “X” is probably not Ci-75 because ci null clones in the A region do not turn on hh at P-type levels [1078, 2832, 2834]. Alternatively, En might turn on hh by using a co-activator, such as Blistered [2729], Extradenticle [3324, 3325, 3861], or the Trx-G chromatin-remodeling complex (cf. Ch. 8) [451]. En is potentially secretable [811, 2085, 2661] but is not used thusly here. Slimb helps keep wg off in the P region (not shown) via a Hh-independent route [2856]. After [2832, 4479, 4539]. See [2834] for further nuances.

112

IMAGINAL DISCS

5 6

4

D A

7

Hh 3

en 3-A

ci 3

dpp 3-P

Dpp 3

omb 3

brk 3

H15 3

hth 3

3

P

8

hh 3

V

2

h P

AN D

1

? ?

ac P

AND

wg E-P

Wg 3

? ? FIGURE 5.8. Sectorial domains of gene expression in the leg disc (which become stripes along the leg) plus one annular domain (hth). The ﬂow of control is from en (upper right), which establishes the A-P axis, through dpp and wg, which polarize the D-V axis, to ac (lower left), which allocates cells to bristle vs. nonbristle fates. Black areas chart mRNA (transcribed from endogenous or reporter gene) or protein levels during embryogenesis (E), late 3rd instar (3), early pupal period (P), or adult (A) stage. Protein levels are given for Hh (estimated based on data from wing discs [4136] ), Dpp (range is not exactly known [1169, 4265]), and Wg [2456] . Shading denotes degree of expression. Areas are mapped onto a disc, although some genes turn on only after 3rd instar (e.g., ac). The span of stages (e.g., “3-P”) indicates when expression has been seen but does not connote conﬁnement to that period. Wiring ( activation; inhibition) illustrates a few genetic interactions (see text or sources cited below). “and” means that both inputs are needed to activate the target gene. Thus, the dorsal hairy stripe is caused jointly by Dpp and Hh (neither alone is sufﬁcient), while the ventral stripe is caused jointly by Wg and Hh [1778]. Interactions between Dpp and Wg (not shown) involve mutual antagonism along the D-V axis and cooperation along the proximal-distal axis (see text). Genes (“DO” = details omitted): ac (achaete; DO: expression areas proximal to the tibia) [3193], brk (brinker) [2049], ci (cubitus interruptus) [1135], dpp (decapentaplegic) [2069, 3122] , en (engrailed) [2954] , h (hairy) [3193] , H15 (a T-box relative of omb) [3762] , hh (hedgehog) [4254] , hth (homothorax) [4760] , omb (optomotor-blind) [2049] , wg (wingless, whose stripe in pupal legs is ∼5 cells wide [1039]) [4277] . All these genes are known to have LOF or GOF effects on pattern, except H15 (whose null phenotype is wild-type [3087]) and omb. Two genes whose expression resembles that of H15 – nkd [4863] and Dfz3 [3977] (not shown) – also have no LOF defects. Enhancer-trap line c409 (not shown) matches ci’s pattern [2684], as does aldehyde oxidase [2340, 4051]. See [489] and FlyView (ﬂyview.uni-muenster.de) [2027] for other enhancer-trap expression patterns. Areas of expression that are not wedge shaped (omitted) include arcs (e.g., GAL30A [487], grain [509], Sex combs reduced [2393] , Ultrabithorax [3397]), parabolas [2977], a spot at the femoral chordotonal organ [1235], etc. [834, 1574, 2684, 4522]. Augmented from [531]. In some specimens the dpp-on and wg-on sectors appear as wide as shown [2954, 4277, 4849] , but often they look narrower [231, 487, 3762] . Likewise, the en domain sometimes displays a central acute angle as shown [2954, 3329] , but it can also seem straight [1472, 4254] or even obtuse [315]. These vagaries arise from variations in folding, viewing angle, and focal plane, as well as from inherent irregularities in the A/P (and other?) boundaries [1833, 4254]. In early pupae, a gap arises between the en domain and the dorsal h sector [1778], although it may not be as large as implied here. N.B.: Hh, Dpp, and Wg only seem to deﬁne D-V sectors of h expression [1778]. Therefore, one remaining mystery is: what factors deﬁne h-on sectors along the A-P axis? Another is: what factors deﬁne the edges of ac-on stripes that are not created by “h ac” inhibition?

CHAPTER FIVE. THE LEG DISC

between V rows 1 and 8 than between D rows 4 and 5 (Fig. 5.1e). Thus, the areas ﬂanking L2&7 may actually be closer in size – a detail that is important in assessing how far Dpp or Wg signals travel. Reduction of dpp or wg function does not just result in loss of structures from one or the other side of the leg. Commonly, the missing structures are replaced by a mirror-image copy of the remaining part (Fig. 5.5) [177, 1812, 3317, 4032]. The V/V dpp LOF phenotype is like a hand with two palms and no back, while the D/D wg LOF phenotype is like a hand with two backs and no palm. Both D/D [560, 1812] and V/V [2082] legs have uneven girths, suggesting that leg segments vary in the fraction of their circumference that is under wg or dpp control. According to the PC Model, such “Janus” oddities should, in principle, be easy to explain. The deﬁciency portion of the deﬁciency-duplication phenotype would be due to tissue loss, and the duplication would arise via juxtaposition of cells with disparate angular coordinates from around the necrotic zone, followed by circumferential intercalation by the shortest route. In fact, however, although necrosis is seen in dpp LOF discs [529, 2739, 2849], it is negligible in wg LOF discs [2015, 2929, 4683]. An even more serious problem for the model pertains to intercalation. According to PC logic, the V/V dpp LOF phenotype arises because loss of the D (dpp-dependent) sector removes ≥50% of the angular coordinates. If so, then the shortest route for row 2 and 7 cells to take when confronted must be the V arc that includes rows 1 and 8. However, the D/D wg LOF phenotype implies a different shortest route (i.e., the D arc encompassing rows 3–6) for the same row 2/7 confrontation. The PC Model is stumped by this paradox. When wg is artiﬁcially expressed in GOF clones on the D side of the leg, the cells in and around the clone make ventrolateral (row 2- or 7-type) bristles [837, 1039, 4159]. This result proved that Wg can act as a ventralizing agent [3923], but it raised the question of why the cells do not become fully ventralized. The failure to induce ventral (row 1- or 8-type) bristles was initially attributed to low output of the wg transgene that was used [4159], but two later ﬁndings revived doubts as to whether Wg is really a morphogen in the leg disc (cf. proof for such a role in the wing disc [4849]): 1. Activating the Wg pathway (cf. Fig. 5.6) downstream of the receptor (via sgg null clones) can induce 1/8type bristles in the very heart of the Dpp domain or anywhere else around the tarsus [1039, 1833, 4666]. This

113

fact refutes the excuse that Dpp is too strong in its own realm to permit V-type states there. Instead, it suggests that Wg only speciﬁes ventrolateral fates, with fates nearer the V midline being speciﬁed by a second ligand (another Wnt? [3919]) that is also transduced via Sgg [4666]. (Similar logic may govern the embryo’s Dpp gradient [2730].) 2. If Wg were a morphogen, then it should specify fates in a concentration-dependent way. However, when the disc is ﬂooded with Wg (via Gal4 drivers that enforce nearly ubiquitous expression of UAS-wg), the V region sprouts a forest of 2/7-type bristles but no extra 1/8-type bristles [4666]. Again, the implication is that Wg merely controls ventrolateral fates. Midventral identities might be dictated by Hh (cf. D rows [2533]), because extra 1/8-type bristles arise when the Hh pathway is turned on in the V area by DC0 LOF (Figs. 3d of [2491] and 6h of [3238]), although row 1 lies in the P compartment (deaf to Hh?) [1800, 2449]. Dpp’s role as a dorsalizing morphogen in the leg is equally dubious. Excess Dpp (UAS-dpp driven by dppGal4) can induce expression of omb (optomotor-blind ) – a marker gene for D identity – in the V region of the leg [487], but overexpression of Dpp within its own domain fails to induce extra (4/5-type) D pattern elements [2954]. The lack of a signiﬁcant GOF phenotype for a gene that has a strong LOF phenotype was seen before in scute (cf. Ch. 3). In that situation, the explanation was a combinatorial interaction between Scute and Daughterless – a dimerization partner that may be limiting under excessScute conditions. Here, too, with Dpp, there could be a simple reason why “shouting” achieves no more than “whispering.” Namely, another signal might be needed in addition to Dpp. Indeed, Hh might be the hidden player in the both the Dpp and Wg stories: {Hh and Dpp} {Hh and Wnt?}

row 4/5-type bristles? row 1/8-type bristles?

Dpp does display some dose-dependent properties (see below) [2954], but they are irrelevant to Dpp’s role as a dorsalizing agent per se. This confusing state of affairs stands in stark contrast to the embryo, where Dpp’s morphogen properties are so well documented in terms of fate speciﬁcation [1281, 3532, 3693, 4058, 4492], gradient distribution [1212, 1927, 2003], and dose sensitivity that is manifest not only in discrete thresholds [127, 1091, 1211, 4613], but also in haplo-insufﬁciency [1872, 1987, 3458, 3693]. It also contrasts with the wing, where Dpp’s role as a morphogen has been well established (cf. Ch. 6) [2455, 3074].

114

Dpp and Wg are mutually antagonistic In 1996, a ﬂurry of articles revealed a mutual antagonism between Dpp and Wg signaling. By manipulating dpp or wg expression directly [487, 2059, 2082, 2954, 4277] or by interfering indirectly with components of their pathways [2059, 3329, 4277], these experiments uncovered a ﬂip-ﬂop circuit that was suspected from earlier clues [1812, 2058]. Evidence that Wg inhibits Dpp is summarized below. 1. Under wgGOF conditions (dpp-Gal4:UAS-wg), where wg is forced to be expressed in the Dpp sector (i.e., dpp-expression zone), the level of dpp transcription decreases [487, 2082, 4277]. 2. In wg LOF mutants, dpp expression increases in the Wg sector (as wg expression decreases) [487]; this substitution is noticeable within 24 h after reducing wg function [4277]. 3. When wg expression is extinguished in the Wg sector (due to dpp-Gal4 activating UAS-dpp along the entire A/P border), expression of dpp-lacZ increases in that V area [487, 2954]. The same effect is seen in Wgsector somatic clones whose Wg pathway is blocked (by dshnull [1833, 2059]). 4. Somatic clones that are both DC0null (which makes cells think they have received a Hh signal) and wg null express dpp when they arise anywhere within the vAC (or dAC) [2059]. Evidence for a reciprocal inhibitory effect of Dpp on Wg is as follows: 1. Overexpressing dpp along the A/P boundary (dppGal4:UAS-dpp) suppresses Wg in the Wg sector [487] except at the disc center (endknob) [2954, 4277]. Concomitantly, expression of the enhancer trap H15-lacZ (a V marker) vanishes in the V region, and omb expression (a D marker) is now detected [487] (see Fig. 5.8 for expression domains). A similar effect is achieved when a constitutively active (ligand-independent) Dpp receptor (tkvGOF ) is used instead of excess Dpp (dpp-Gal4:UAS-tkvGOF ) [4277]. 2. When the level of dpp transcription decreases in the Dpp sector (due to dpp-Gal4:UAS-wg), the endogenous wg (distinguishable from the UAS-wg transgene by a lacZ insert at the wg locus) is activated [487]. (Concomitantly, H15-lacZ expression appears and omb expression disappears [487].) Upregulation of wg also occurs (although only rarely) when dppnull clones reside in the Dpp sector [2059] or when Dpp transduction is blocked in dorsal somatic clones (by puntLOF [3329] or tkvLOF [2059, 3329]).

IMAGINAL DISCS

3. In dppLOF mutants, Wg is now detected in the Dpp sector (as dpp expression decreases) [487]. The ectopic Wg is restricted to the tarsal region – the only area converted to V identity in these hypomorphic genotypes [1812]. A similar substitution of wg for dpp expression occurs when the Dpp pathway is blocked ubiquitously (by puntLOF [4277]), but in that case wg is transcribed in the entire dorsal sector [4277]. 4. Somatic clones that are both DC0null and dppnull express wg when they arise anywhere within the dAC (or vAC) [487, 2059]. Similar effects are seen when DC0null is coupled with sggnull , which inhibits dpp [2059]. In wild-type discs, Dpp’s ability to suppress wg transcription must be greater than Wg’s ability to suppress dpp transcription because dpp is transcribed at a low level in the Wg sector, whereas wg is not transcribed at all in the Dpp sector [231, 2466]. Dpp has been shown to also block Wg’s downstream effects: when the same group of (dorsal femur) cells is forced to express both dpp and wg (by using 30A-Gal4 to drive UAS-dpp and UAS-wg), wg is unable to activate its puppet gene H15 [487]. Whether Wg can similarly block Dpp’s downstream actions is not known. The Dpp-Wg antagonism means that the D and V sectors are each like a seesaw that must tilt one way (Dpp) or the other (Wg), but it does not elucidate why each sector tilts a speciﬁc way. The answer may be found in the embryo [487, 3862]: the nascent leg disc incorporates part of an ectodermal Wg stripe as its V sector (Fig. 4.4b), and henceforth Wg predominates there [880]. Why the D sector tilts to a Dpp state is unclear, but abundant evidence (see below) indicates that the alternative (i.e., two symmetric Wg sectors) is incompatible with distal outgrowth. The mutual exclusivity of Dpp or Wg activity may sharpen the boundary between their domains so that other circuits can come into play [2954]. Oddly, ventral suppression of dpp also requires Notch during 2nd and early 3rd instars [886]: “{N and Wg} dpp”. Notch enforces a comparable bipolarity in wing discs (wing vs. notum; cf. Fig. 6.9) [886] and eye discs (eye vs. antenna) [2353, 2362]. The ability of dpp-Gal4:UAS-wg to convert the D side of the leg into a purely V phenotype (with attendant H15-lacZ [487]) has been construed as evidence that Wg can specify midventral identities after all [2082], despite the skepticism voiced above. However, the objection raised by the sgg null clones still stands because they fully ventralize without “cheating” (i.e., using the mutual-antagonism circuit to cripple the opponent

CHAPTER FIVE. THE LEG DISC

(Dpp) prior to specifying cell fates). Another attempt to resurrect Wg as a leg-disc morphogen was based on a dose dependence of Wg’s effects on the D side of the leg. With increasing dose, Wg successively (1) removes D structures, (2) induces leg-to-wing homeosis (cf. Ch. 8), and (3) achieves total repression of dpp, whereupon the D side vanishes and is replaced by a V duplicate [2082]. Again though (as with dpp), none of the thresholds is really pertinent to whether Wg is a morphogen along the D-V axis. What is needed is proof that greater doses of Wg directly induce increasingly ventralized bristles, but no such evidence exists [3087].

115

3.

4.

Dpp and Wg jointly initiate distal outgrowth In the Boundary Model, distal outgrowth is supposed to be triggered by a combination of morphogens [2804]. Given that the leg tip comes from the disc center, which is the sole point of contact between the Dpp and Wg sectors, it was reasonable to suppose that the combination of these two diffusible signals might jointly cause outgrowth [617, 4490]. This “{Dpp and Wg} distalize!” imperative was proposed by Campbell et al. [620] in 1993. In 1994, the upstream step “Hh {Dpp and Wg}” was added by several teams (Short-range Inducer Model) [231, 1037, 2466]. Thus was born the “Hh-Dpp-Wg Model” for distal outgrowth (Fig. 5.4c), which invoked a dependence of the proximal-distal axis on the D-V axis: Hh

{Dpp and Wg}

5.

distalize!

Many proteins in the TGF-β [63, 2733] and Wnt [2261, 3150, 3910] families act as growth factors, and the data below argue that Dpp and Wg also do so. Some of these experiments use Distal-less (Dll) and aristaless (al) as indicators for prospective distal tips. Both Dll and al are homeobox genes [836, 3798]. Dll is expressed in the disc’s tarsal region (plus trochanter and tibia) [881, 1037, 1561], while al is expressed where the claws arise (plus dorsal coxa and ventral tibia) [620, 881, 3798]. The facts below support the Hh-Dpp-Wg Model: 1. Ubiquitous expression of Hh expands the solid circle of Dll into a broad band straddling the dAC/vAC border (as Dpp and Wg spread to ﬁll dAC and vAC) [1037] and similarly stretches the Al spot into a stripe (Fig. 5.9c) [617]. Both responses imply a combinatorial “{Dpp and Wg} {Dll and al}” control mechanism. 2. Ubiquitous expression of Dpp extends the Dll circle into the ventral sector where Wg signal is naturally received (as per a “{Dpp and Wg} Dll” rule)

6.

and provokes overgrowth in that same area [1037]. Outgrowths from the Wg sector (along with an extra Al spot) are also induced when dpp-Gal4 drives UAS-dpp in that region [2954]. Ubiquitous expression of Wg lengthens the Al spot into a stripe along the Dpp sector (Fig. 5.9d) [620] (as per a “{Dpp and Wg} al” rule), and concentric oval folds (indicating overgrowth) form around this stripe. The same effect (plus overproliferation of dorsal cells) is seen when Wg is misexpressed in the Dpp sector (dpp-Gal4:UAS-wg) [4666]. hhGOF clones only induce outgrowths when they straddle the dAC/vAC border (where both Dpp and Wg would be induced) [231, 617], whereas AC hhGOF clones that miss the border cause partial AC duplications and local overgrowth (cf. Fig. 6.6) but no distal outgrowths. The same is true for DC0LOF [2058, 2491, 3238] or slimbLOF clones [2060, 4279], although the outgrowths tend to be distally incomplete [2533]. Such boundary dependence recalls the Boundary Model (cf. the paint-spattering scenario). dppGOF clones only induce outgrowths ventrally [1037, distalize!” imperative. 2059] as per a “{Dpp and Wg} The branches are typically stunted (1–2 segments long), but truncation could be due to low output of the transgene. Longer branches are seen when the endogenous dpp gene is turned on along the ventral A/P border (via the Dpp-Wg antagonism circuit) in somatic clones whose Wg pathway is turned off (by dshnull [1833, 2059] or by the compound DC0null wg null [2058, 2533]). wg GOF clones only induce outgrowths when they reside along the Dpp sector as per the “{Dpp and Wg} distalize!” rule [4159], in which case they induce ectopic Al [617, 620] and Dll [1037]. Similar effects are seen when clones in this area have a hyperactive Wg pathway due to intrinsic malfunction (by sggnull [1039, 1833, 2059] or dAxinnull [1698]) or a seesaw response (Dppdown causes Wg-up) to blockage of Dpp signal transduction (by puntLOF [3329] or tkvLOF [2059, 3329, 4277]). Because sggnull clones induced after mid-2nd instar fail to trigger outgrowths [4666], such clones may need to exceed a certain size to “knock out” the dpp-on target area and tip the seesaw. A bizarre twist on this story may explain in situ duplication of 3rd-leg discs in lethal (2) giant discsLOF (l(2)gdLOF ) overgrowth mutants. Over a period of several days, a tongue of Wg expression arcs around anteriorly to meet the Dpp sector, where an extra disc then arises [558], but duplication is prevented when l(2)gdLOF ﬂies are also made homozygous for dppLOF or wg LOF .

116

IMAGINAL DISCS

en

Hh

"dAC"

dpp

"vAC"

wg

al

a b "dAC"

Wild type

? and

Hh

dpp

?

Dpp

and and

En

wg

and

Al

and

Al

Wg

"vAC"

?

c

?

ars later nst i 2

Ubiquitous Hh

"dAC"

? and

Hh

dpp

?

Dpp

and and

En

wg

Wg

"vAC"

? ? 2 in

d

?

e ?

Dpp

Ubiquitous Wg Wg

stars later

Wild type ?

?

Dpp and

?

and "dAC"

Al and "vAC"

Wg

?

?

?

CHAPTER FIVE. THE LEG DISC

Given the “{Dpp and Wg} al” link and Al’s expression in the claw region, it is odd that no extra claws arise when the Al spot widens as a result of ubiquitous Hh or Wg [4666]. Conceivably, other factors elicited by “Dpp and Wg” may be suppressing extra claws (e.g., a central spot of mitotic quiescence [1599, 1811] that expands along with Al [4666]). Equally perplexing is why Al is expressed at the inception of the disc (in a pattern similar to Dll) and during 3rd instar [620, 881, 1304], but not during the intervening 1st and 2nd instars. One last riddle about Al is: why do outgrowths at the dAC/vAC interface (e.g., due to DC0 null clones) tend to be distally incomplete [2533], because they should express Al and other distal markers? Indeed, al GOF clones fail to induce leg duplication at a signiﬁcant frequency [620]. Attempts to ﬁgure out where al acts within the distalization circuitry were stymied until recently by the lack of a null allele [620, 4682]. In 1998, it was shown that al function is not needed for distal leg development [618], so al must act downstream. These quibbles about Al are minor compared with the issues about Dpp.

117

The presence of endogenous dpp mRNA in the Wg sector was initially problematic for this model because distalization should occur wherever Dpp and Wg signals are both received [617, 620]. What prevents extra tips from growing out of the V region? Evidently, Dpp’s concentration is insufﬁcient to let it interact with Wg to trigger outgrowths. When the amount of Dpp is increased (via dpp-Gal4:UAS-dpp), extra tips indeed emerge [2954]. When the Dpp level is raised still farther (by using other strains or temperatures), the V sector virtually disappears due to suppression of Wg. These results indicate that the threshold for Dpp-Wg antagonism (“Ta ”) is higher than the threshold for Dpp-Wg cooperation in distalization (“Td ”) [2954]. Another threshold is evident in the prospective claw area (“Tc ”): slight reductions in Dpp signaling prevent claws from forming (but legs are otherwise normal) [4033], and a tongue of Wg extends into the dorsal endknob (where claws arise) as Dpp expression retreats [2954]. At the highest level of Dpp expression achieved by dpp-Gal4:UAS-dpp, a ﬁnal threshold (“Tt ”) is seen: the only vestige of the Wg wedge that still

FIGURE 5.9. Logic of the Hh-Dpp-Wg circuitry that governs leg patterning (cf. Fig. 5.4c).

a. Regions (cf. Figs. 5.1c, 5.8, 5.11) where key genes are transcribed (en, engrailed; dpp, decapentaplegic; wg, wingless; al, aristaless), where protein is detected (Hh, Hedgehog), or where discrete types of competence prevail (“dAC” vs. “vAC” are dorsal and ventral parts of the anterior compartment). Shading or hatching symbol is shown under each name. b. Core circuit (cf. Fig. 2.7 for symbol key), whose logic is as follows. Like en, hh is transcribed in the posterior compartment (PC), but unlike En, Hh protein diffuses some distance into the AC. PC cells are “deaf” to Hh signal because they express En (cf. Fig. 6.4) [4229], but AC cells within Hh’s range can respond. Hh’s diffusion range in the AC is depicted as two opposing sectors (after the ﬁrst “and”). Cells in the dAC and vAC areas respond differently to Hh: dAC cells transcribe dpp, while vAC cells transcribe wg. Dpp and Wg (like Hh) are diffusible signals, but their ranges (graded shading) and activity levels are uncertain. Cells at the center (where dpp and wg sectors touch) make Al. From this diagram, it would seem that Dpp and Wg need Hh continually, but in fact, turning hh off during the last day of larval life causes no notable defects aside from missing claws [1472]. c. Evidence for the “Dpp + Wg = Al” circuit. Ubiquitous expression of Hh during mid-late 3rd instar expands dpp and wg transcription into the entire dAC or vAC [231, 2466] (assessed by lacZ reporters). As a result, cells bordering the dAC/vAC boundary are exposed to the same high-Dpp, high-Wg conditions as cells at the center. Hence, the Al spot extends into a stripe along this line. When hh is turned on (transiently) during 1st instar, discs become grossly misshapen, and Dpp antibody (above) or a wg reporter-gene (below) reveals that Dpp and Wg continue to be expressed, hence suggesting that each acts as a mitogen within its own (dAC or vAC) territory. The PC remains normal in size (despite a ∼3-fold larger AC), while the Al stripe elongates to span the enlarged AC (not shown). d. Further evidence for the “Dpp + Wg = Al” circuit. Ubiquitous expression of Wg causes the Al spot to lengthen into a stripe along the Dpp sector. e. Mutual antagonism between Dpp and Wg. The act of synthesizing Dpp apparently precludes synthesis of Wg within the same cell, and vice versa. AC cells appear to adopt different states of competence based on whether they receive a Dpp or a Wg signal: cells that receive Dpp (dAC plus “?” sector) remain competent to make Dpp in response to Hh, while cells that receive Wg (vAC plus “?” sector) remain competent to make Wg in response to Hh. This “seesaw” can be forced the wrong way by (1) blocking Dpp [1811, 1812] or its transduction [753, 3329, 3932, 4277] in D cells, (2) blocking Wg [177, 880, 1673, 1811, 2110] or its transduction [519, 2262, 3317] in V cells, (3) forcing D cells to make Wg [487, 2082, 4277], or (4) forcing V cells to make Dpp [487, 2954, 4277]. Data for panel a comes from refs. in Figs. 5.8 and 5.11. Sources for other panels are b [231, 620], c [231, 617, 2466], d [620], and e [1811]. N.B.: When Wg is ubiquitously expressed (d) during 1st instar, discs also deform (not shown) but do not grow as much as with excess Hh (c) [617]. Distal-less behaves like Al (c) but in a broader area [1037]. Ventral sector of dpp transcription is omitted (see text), as are coxal and tibial regions of Al expression [620].

118

expresses Wg is the tip (just ventral to the claw spot) [2954, 4277]. In summary, the following events occur in sequence as the amount of Dpp rises from a slightly subnormal level: (1) Dpp displaces Wg from the claw area, (2) an extra distal outgrowth arises in the V region, (3) Wg totally vanishes except at the tip of the Wg sector (i.e., Tc < Td < Ta < Tt ).

IMAGINAL DISCS

3.

But Dpp seems more crucial than Wg as a growth factor The experiments listed above prove that Dpp and Wg are sufﬁcient for inducing extra distal tips, but they do not address whether both are necessary. The ability of wg LOF legs (branched or unbranched) to fully distalize [177, 880, 1812, 3317] raises the question of whether Wg is needed. Such legs can be D/D symmetric from the sternopleura to the claws with only a slight change in leg length [177, 519, 1812, 3317, 4278], while V/V dpp LOF legs (which express wg on both sides) are severely stunted [2082, 4277]. Likewise, when wg is transiently silenced before 1st instar (12–24 h AEL), 60% of the D/D legs distalize fully (vs. 80% when wg is stiﬂed for 12 h in early 3rd instar) [880]. These effects are not just due to leakiness of the LOF alleles used because wg null clones (induced in 1st instar) can also yield distally complete (albeit only partly D/D) legs [1811]. Other clues also point to an inequality in the roles played by Dpp and Wg: 1. Unlike (distally complete) wg LOF legs, dpp LOF legs tend to lose distal elements as a function of the strength of the LOF allele [529, 1812, 4033]. In fact, dpp was once thought to primarily govern the proximaldistal axis (instead of the D-V axis) for this reason [33, 834, 1427, 4675, 4682]. 2. Janus phenotypes are manifest by both wg LOF (D/D) and dpp LOF (V/V) legs, but dpp LOF legs can also exhibit a “V/-” phenotype where the deﬁciency is not associated with a duplication [1812]. In such cases, the D side of the tarsus is missing but is not replaced by V-type tissue (Fig. 5.5d). No analogous “D/-” defects are seen in wg LOF legs. Why? Unlike Wg, Dpp may not only be serving as a morphogen, but also as a trophic survival factor [4080]. Among the facts that support this conjecture are (1) dpp LOF discs display appreciable apoptosis [529, 2739, 2849], whereas wg LOF discs manifest relatively little [2015, 2929, 2932, 4683]; (2) the tissue loss and poor growth of dpp LOF wing discs are rescuable by coculturing with wild-type discs, implying a diffusible anti-apoptosis factor [529]; and (3) apoptosis in wing and leg discs is triggered (via the JNK pathway [971]) when genes in the Dpp pathway are suppressed [12],

4.

5.

6.

7.

whereas milder wg LOF effects occur via omb. Dpp’s link to apoptosis may be at its receptor because Tkv binds proteins that inhibit apoptosis [3170]. Unlike wg LOF legs, which commonly branch [177, 1812, 3317, 4278], dpp LOF legs bifurcate only rarely [529] and when they do, the side branch dwindles distally (L. Held, unpub. obs. of 60 dppd2 legs). Evidently, branches easily grow when the seesaw circuit causes dpp to be derepressed (e.g., in V clones that are dshnull [1833, 2059, 2262, 4278], DC0null wgnull [2058, 2533], or dAxinGOF [1698]), but dorsal outgrowths are pitifully small by comparison when wg is derepressed (e.g., in D clones that are punt LOF [3329], tkvLOF [2059, 3329, 4277], or sgg null [1039, 1833]). This disparity may partly be due to Dpp’s greater ability to snuff out Wg nearby than vice versa [1833, 2059, 4277]. A similar imbalance is seen when comparing dppGal4:UAS-dpp legs to dpp-Gal4:UAS-wg legs: the former (excess Dpp) often have outgrowths from the V surface [2954], whereas the latter (excess Wg) rarely display outgrowths from either side [2082, 4666]. Why then can wgGOF clones induce outgrowths on the D side [4159]? Perhaps because the ones that arise near, but not in, the Dpp sector spark a “Dpp + Wg” reaction without downregulating dpp. When wgGOF (or sggnull ) clones reside laterally (far from the Dpp sector), they evoke only tiny outgrowths [1039, 1833], indicating that Wg alone is a poor mitogen. Distal outgrowths can be induced ventrally by doubly mutant DC0null wgnull clones (which express Dpp), but no outgrowths (dorsal or elsewhere) are seen for DC0null dppnull clones (which express Wg) [2058, 2533], implying a greater role for Dpp than Wg in distal outgrowth. Frequencies and sizes of somatic clones are reduced when the clones are prevented from transducing Dpp (by puntLOF [3329] or tkvnull [3329, 4277]) but not when they are prevented from transducing Wg (by dshnull [2059]). This rule is broken by arm (armLOF clones die if they arise ventrally or distally [3317]), but Arm plays a vital role (as a component of junctions) aside from signaling. Discs whose whole A/P border is dominated by Dpp (dpp-Gal4:UAS-dpp) widen perpendicularly to the border [487, 2954, 4277] (by expanding the A compartment?), whereas discs whose A/P border is ruled by Wg (dpp-Gal4:UAS-wg) lengthen along the A/P line instead [487, 2082, 4277].

Dpp cannot be the sole agent in distalization because tips would arise all along the D midline. Thus, if Wg is

CHAPTER FIVE. THE LEG DISC

superﬂuous [4666], then another agent “X” must overlap Dpp centrally (see the argument above for why wg GOF does not fully ventralize cells) and the rule must be “{Dpp and X} distalize!”. (X might override the DppWg antagonism to enforce the Tt threshold [2954].) As for why ectopic Wg triggers extra tips, the leg disc circuitry must have a “Wg X” link, but other factors may redundantly keep X active centrally [2533]. Heating t.s. wg LOF mutants during 2nd instar abolishes Dll centrally and causes truncations [1037] like what happens when discs are deprived of Dpp or Hh. However, shift studies with t.s. alleles show that neither Wg nor Dpp is needed for distalization after early 3rd instar (although they are still required for the D-V axis) [883]. The conjecture that Dpp is more important than Wg for distalization would help explain two enigmas from the Cellular Epoch: the “Dorsal Remnant” and “Triplications” Mysteries (see the start of this chapter). When cells are randomly killed in a young disc, whole sectors can vanish. Judging from the structures made by surviving cells, virtually all parts of a disc except its Dpp sector are dispensable (Fig. 5.2b) [3449]. The reason may be that a disc’s growth factors come mainly from there. Identical D-remnant legs can be produced by overexpressing Dpp in the Wg sector (which then fails to develop due to “Dpp wg”) [2954]. The Triplications Mystery [1495] also implies an asymmetry of mitogenic function along the D-V axis. Perhaps, D patches of apoptosis that overlap the Dpp sector reduce the Dpp level so much that D outgrowths starve for mitogens and hence converge, whereas V patches of apoptosis have no such effect and thus allow V outgrowths to diverge. The ability to elicit convergent vs. divergent outgrowths by nicking D vs. V sides of cockroach legs [384, 547, 2804] reveals a similar asymmetry that is consistent with this argument. These etiologic scenarios are seemingly contradicted by the fact that dpp upregulates, rather than downregulates, when discs undergo widespread cell death [489, 2856], and indeed, apoptosis is induced by the same t.s. allele of suppressor of forked that causes triplications [1496, 1502]. However, too much Dpp has been shown to reinforce apoptosis as severely as too little Dpp (in wing discs) [12], so this objection may be unwarranted. One fact at least is certain: the dpp-on and wg-on sectors respond differently to trauma. The dpp-on sector broadens, whereas the wg-on sector is unaffected [2856]. This asymmetry probably holds the key to both mysteries, although important links in the connecting circuitry remain obscure.

119

If Dpp is the major mitogen in a leg disc, then it should be able to reach every cell. At one time it was thought that cells on the V side of the disc might receive sustenance from the low level of Dpp in the Wg sector [2954], but ventral dpp expression can be eliminated (via dpp null cell clones) without any detectable effect on growth or patterning [1811]. If ventral cells must depend on dorsally produced Dpp, then the question of Dpp’s diffusion range becomes pivotal (see below). The mitogenic capacity of at least one element in the Hh-Dpp-Wg circuit is made obvious by a grotesque deformity seen when hh is transiently turned on ubiquitously in 1st-instar discs (Fig. 5.9c) [617]. Under these conditions, dpp and wg are transcribed throughout dAC and vAC, respectively, but oddly (given the Hh-Dpp-Wg Model) there seem to be no outgrowths at the dAC/vAC boundary. The AC of these discs broadens tremendously, becoming ≥3 times wider than the P compartment by the 3rd instar. Because dAC and vAC expand equally, it would seem that Dpp and Wg must both be mitogens, but each might be suffusing the whole disc. When Wg alone is overexpressed via dpp-Gal4:UAS-wg, some excess growth occurs [4666], but it is meager by comparison. Overexpressing Dpp via dpp-Gal4:UAS-dpp is also relatively ineffectual in provoking extra growth in its own domain [2954], implying that a Dpp threshold for hyperplasia is crossed in one (hh GOF ) but not the other (dpp GOF ) experiment. Another peculiarity of the Hh-Dpp-Wg circuitry is seen with DC0 null clones, which commonly cause distal outgrowths but are prevented from doing so when they are also null for dpp or wg [2533, 3238]. This result is heretical because the (Wg-expressing) DC0 null dpp null clones should have induced outgrowths whenever they were near the dpp-on sector, and the (Dpp-expressing) DC0 null wg null clones should have induced outgrowths whenever they were near the wg-on sector.

The A/P boundary can migrate when its ‘‘jailors’’ are ‘‘asleep’’ When Wg is forced into the Dpp domain (via dpp-Gal4: UAS-wg [4277] or ptc-Gal4:UAS-wg ts [2082]), it causes a mysterious shift in the boundary that separates en-on from en-off cells: this boundary migrates anteriorly, but only in the dorsal half of the disc where dpp is downregulated (Fig. 5.10d): en is normally expressed in cells of the posterior compartment of each disc. In 15◦ C ptc-Gal4:UAS-wgts discs [the permissive temperature for wgts ], en expression has broadened into anterior cells. . . . In leg discs the en expansion is primarily restricted to dorsal cells and occurs in approximately 80%

120

IMAGINAL DISCS

en

dpp

Hh

wg

a b Wild-type

dpp

c.e.

p.m.

or Hh

p.m.

and

wg

hh

En

Hh

and

c.e.

En

c

contact

Wild-type

1/4 t p.m.

t

c.e.

etc.

c.e.

P

c.e.

Regeneration

LOF

hh p.m.

c.e.

A

contact t

t

etc.

A

3/4

P P p.m.

d

c.e.

c.e.

c.e.

e

dpp-Gal4:UAS-wg

dpp-Gal4:UAS-dpp t

t

Duplication

t

t

Loss of dorsal barrier

Loss of ventral barrier

CHAPTER FIVE. THE LEG DISC

(n = 60) of leg discs. The anterior en expression is dependent upon ectopic Wg. . . . However, anterior en-expressing cells do not take on posterior fates in the adult. . . . The mechanism behind the en expansion is not clear. [2082]

A similar phenomenon was independently discovered when Dpp is forced into the Wg domain (via dppGal4:UAS-dpp) [2954, 4277]: the en on/off boundary shifts anteriorly, but only in the ventral half of the disc where wg is downregulated (Fig. 5.10e): In addition to the reduction in ventral-anterior wg expression, the leg discs with increased dpp expression exhibited an altered pattern of en expression. Cells expressing en were present across the region that would normally make up the ventral-anterior region of the leg disc. The mechanism for this pattern change is not known. We propose that the increased expression of dpp along the ventral A/P boundary reduces wg expression and that this destabilizes the A/P boundary. Two mechanisms seem plausible. In one, the destabilized A/P boundary migrates across the anterior-ventral region of the disc and as the boundary passes, cells begin to express en. Ectopic hh overexpression in the A compartment can induce en expression [1647]. We postulate that expression of wg in the anterior-ventral [area] normally prevents cells on the A side of the boundary from inducing en expression in response to hh from the P compartment. However, when increased dpp reduces the level of wg expression in the anterior-ventral [area], the cells on the A side of the boundary might respond

121

to the hh coming from the P compartment by activating en expression. This in turn would permit these cells to induce hh expression, which could signal en expression to occur in the next most adjacent anterior cells. This would result in a processive movement of the compartment boundary across the anterior-ventral region that is normally blocked by wg. This proposed migration is reminiscent of the processive migration of the morphogenetic furrow across the eye imaginal disc, a process which is also blocked by wg expression [4390]. [2954]

As the above quote asserts, the correlation of boundary migration with reduced dpp or wg transcription may be causal. To wit, cells that express either dpp or wg may be unable to turn on en in response to Hh. Because Hh activates dpp or wg in each A cell that it reaches (as per the Hh-Dpp-Wg Model), it behaves like a minotaur that builds its own cage. If the cage dissolves due to insufﬁcient Dpp or Wg, then the en-on state can invade the A compartment. The wavefront ratchets itself stepwise by a Hh-En-Hh feedback loop (Fig. 5.10b; cf. the eye [571, 1784, 4169]): Step 1.

Hh diffuses ahead of the hh-on (= en-on) territory and turns on en. It is this “Hh en” link that appears to be disabled by Dpp or Wg. This circuit element is dormant until late 3rd instar [350, 364, 3177, 4228] when it

FIGURE 5.10. Hh-En-Hh feedback loop and how it solved the Quadrant Regeneration Mystery.

a. Regions where key genes are transcribed (en, engrailed; dpp, decapentaplegic; wg, wingless) or protein (Hh, Hedgehog) is detected. Symbols (shading or hatching) are below names. b. Hh-En-Hh loop (cf. Fig. 2.7 for icons). The logic begins at left with 4 maps of a 1st-leg disc: upper vs. lower are Hh vs. En; left vs. right are peripodial membrane (p.m.) vs. columnar epithelium (c.e.). In the p.m. of 1st-leg discs, en and hh are transcribed in a dorsal spot [1472]. In the c.e. (of all leg discs), en and hh are expressed in the P compartment, and Hh diffuses into two sectors (map below ﬁrst “and”) where en-off (A-type) cells can “hear” it (P cells are “deaf”). In theory, these cells should respond by turning on en because a “Hh en” link emerges in late 3rd instar (cf. Fig. 6.4) [1472, 4136], and indeed a sliver of cells within these sectors does so (not shown) [350]. However, remaining cells in the border zone cannot because (1) Hh causes them to express Dpp or Wg (cf. Fig. 5.9b), and (2) Dpp or Wg blocks this “Hh en” step. Thus, the En map (below second “and”) does not change perceptibly. In this steady state (remainder of the loop), En continues to activate hh, and Hh continues to diffuse out of the P compartment. c. As shown in the side-view schematic (lower left), the leg disc is a ﬂat sac (cf. Fig. 5.1b): p.m. cells (upper layer) are squat, whereas c.e. cells (lower layer) are tall. (Elsewhere layers are drawn side by side.) After a cut, both tissues heal together (“contact”), at least brieﬂy. If the cut bisects the en spot, then Hh diffuses (via contact) from p.m. to c.e., where it engenders some en-on cells. (Remaining drawings depict c.e. only.) Such interactions should occur in both 1/4 (upper series) and 3/4 (lower series) pieces. In the former, the en-on patch leads to regeneration (by restoring A-P duality), whereas in the latter it leads to duplication (by instigating a second P compartment). Without Hh activity (hh LOF arrow), the 3/4 piece fails to acquire an extra P domain, so it regenerates instead, as do 3/4 pieces of wild-type 2nd- or 3rd-leg discs, which lack an en spot [1472]. “t” denotes time periods on the order of hours (after healing), whereas “etc.” connotes an extended growth period of several days. d, e. Invasion of the A compartment by the En wavefront (arrowhead) when the Hh-En-Hh loop is unleashed. In these cases, the Dpp-Wg antagonism (cf. Fig. 5.9e) is used to downregulate dpp dorsally (d) or wg ventrally (e). Domains in a are from sources listed in Fig. 5.8. The dpp sector is rendered narrower (cf. Fig. 5.9) so that the ectopic en patch (c) can ﬁt into a dpp-off part of the 3/4 piece, because otherwise the circuit will not work. (Distance from vertical cut line to dpp sector is not known exactly.) Circuitry in panel b is deduced from data in [1472, 2082, 2954, 4136]; c is adapted from ﬁgures in [1472]; and d and e are based on pictures in [2082, 2954, 4277], whence wavefront stages are inferred.

122

Step 2.

Step 3.

IMAGINAL DISCS

prods the en on/off line to creep a few cells ahead of the A/P compartment boundary (Fig. 6.7d) [78, 642, 2832, 3818, 4136]. As a result of Step 1, the en-on edge has stepped ahead of the hh-on edge. The newly created en-on “border” cells should then activate hh via an “en hh” link that was used in the embryo [3747, 4227]. This link should not work in ci-on cells [2992] because hh is more sensitive to repression by Ci-75 than to activation by Ci-155 [2980], so removal of Dpp or Wg must somehow derepress it. (See [703] for how a promoter can be wired for such inputs.) At this stage, the hh-on edge catches up with the en-on edge. As soon as the border cells (with en and hh both on) begin making Hh, the new Hh will diffuse and rekindle the loop at Step 1. (A direct “Hh hh” shunt is precluded because hh is insensitive to Ci-155 [2980].)

This loop can be sparked by ectopic hh GOF virtually anywhere in A compartments of wing [998, 1647, 3739, 4136] or leg discs [1472] (cf. tergites [2436]), but again the en-on state (due to “Hh en”) does not spread far beyond the hh GOF area (clone or region) because of the “Minotaur Scenario.” That is, the hh GOF tissue creates a cage around itself (Hh {Dpp or Wg}) that breaks the feedback loop ({Dpp or Wg} {Hh en}). Nevertheless, any dpp expression that is thereby evoked could sustain the area’s growth and thus amplify the response, as if the minotaur henceforth grows by feeding itself rather than by escaping (cf. Fig. 6.6). In theory, Dpp and Wg could be interfering with a different step (i.e., {Dpp or Wg} {en hh}). However, this possibility seems less likely because the dppon zone remains an effective barrier even when polyhomeotic’s blockage of “en hh” at the A/P border is alleviated [2728]. Oddly, in that case, the en-off edge migrates posteriorly.

Regeneration is due to a Hh spot in the peripodial membrane The observations quoted above were made in 1996. In 1999, Matt Gibson and Gerold Schubiger in Seattle invoked a similar scenario [1472] to explain the same Quadrant Regeneration Mystery that, poetically, Schubiger ﬁrst articulated in 1971 [3808]. As mentioned at the outset of this chapter, that mystery had bafﬂed theorists for decades [354, 1807]. How could one quarter of a disc regen-

erate the other three quarters? What is so special about the upper medial (UM) quadrant? To accommodate this odd behavior, the PC Model had imagined a warped coordinate system where more than half the leg’s angular values are crowded into the UM area. However, this ad hoc assumption undermined the model’s conjecture that a disc stops growing when the density of coordinates attains a preset limit [546, 1301, 1303]. How could the UM quadrant end up with a higher density? The Boundary Model was even more bedeviled because the tip morphogen that it demanded for outgrowth is only made when several compartments convene. How could the UM quadrant, which lies entirely in the A compartment [4076], regenerate distally (Fig. 5.3j) [2804, 2808]? Equally puzzling was why certain fragments that are composed of both A and P cells should duplicate [354]. The key to solving the mystery was Gibson and Schubiger’s discovery of a Hh spot in the peripodial membrane of the 1st-leg disc [1472]. Like the other discs, each leg disc is a ﬂat hollow sac with two surfaces (Fig. 5.1b). The thick columnar layer is separated from the thin “peripodial” layer by a ﬂuid-ﬁlled lumen, and columnar cells are packed ≥10x more densely than squamous peripodial cells [2862, 3426]. The peripodial side has usually been ignored in model building [1008] because it makes so few cuticular structures [526, 2862]. Nevertheless, its cells can interact with the other layer when wound edges heal together after surgery [3564, 3565], and this interaction turns out to be pivotal for leg disc regeneration. One of the two cuts that liberates the “1/4” piece passes through the Hh spot (Fig. 5.10c). When this peripodial edge adheres to the columnar edge, some of the Hh evidently diffuses into the columnar epithelium because a few of its cells begin expressing en (due to the “Hh en” link) and later hh (due to the “en hh” link). The activation of en can be blocked by using a t.s. hh LOF allele to turn hh off during this period (thus disabling the “Hh en” link). Spreading of the en-on state should cease due to the Minotaur Scenario (Hh Dpp {Hh en}), but the few en-on cells that are thus created are evidently enough to “seed” the columnar layer with a new P compartment because the 1/4 piece regenerates circumferentially and distally. The same sort of interaction occurs in the “3/4” piece, but its extra P compartment leads to duplication instead (in P/A/P symmetry), thus obeying the Reciprocity Rule that one fragment regenerates while its complement duplicates (cf. Ch. 4). In both cases, subsequent growth at the wound site is probably sustained by the new dpp-on zone (Hh dpp),

CHAPTER FIVE. THE LEG DISC

regardless of whether the peripodial and columnar surfaces later detach from one another (as they often do [3564, 3565]). Other facts support this hypothetical sequence of events [1472]: 1. Whereas the 1st-leg disc has an appreciable Hh spot (20–30 cells), the 2nd-leg disc does not (0–5 cells). Consistent with the above argument, the UM quadrant of the 2nd-leg disc behaves like that of a hhLOF 1st-leg disc: it typically fails to regenerate. First legs are peculiar insofar as the left and right discs are conjoined [377, 3426] (cf. eye [1777] and genital discs [540]), and it is conceivable that the Hh spot is a vestige (and/or mediator) of that fusion event [1472]. 2. In contrast to the 3/4 piece of the 1st-leg disc (which duplicates), the 3/4 piece of the 2nd-leg disc regenerates. Its Dpp and Wg signaling centers evidently sufﬁce for this “Type 2” kind of regeneration (where A and P compartments are both present at the outset) vs. the “Type 1” regeneration exhibited by 1/4 pieces of the 1st-leg disc (where only the A compartment is initially present). The 3/4 fragment of the 1st-leg disc can likewise be coaxed to regenerate (instead of duplicate) by using a t.s. hhLOF allele to silence all hh activity during the postsurgical growth period. The implication is that Hh itself is not needed for Type 2 regeneration (cf. fragments that can duplicate without an A/P boundary [354]). 3. The idea that a few en-on cells can spark a discwide duplication may seem farfetched, but clonal analyses show that the new halves of duplicated legs can indeed arise from as few as 5–10 cells [1472, 1503]. Moreover, tiny hhGOF clones have a similar Davidvs.-Goliath power: they can cause huge wing duplications (cf. Fig. 6.6). Even colossal changes such as homeosis and transdetermination may stem from small errors in diffusible signals (e.g., Hh) because they are nonclonal in origin [1405, 3445] (cf. Ch. 8). However, not all the details of this process have been worked out. For example, both Dpp and Wg must supposedly be present for distal outgrowth to occur, and an incursion of Hh into the dAC (as described above) should only induce Dpp, not Wg. Nevertheless, Wg is detected in precisely this wound area [1472]. What circuit activates Wg in the dAC? Another question concerns the endogenous Hh spot itself. If the same “Hh en” link that is awakened in the columnar epithelium is also activated in the peripodial layer in late 3rd instar, then what prevents this spot from expanding (via the Hh-En-Hh loop)? Is there a “cage” comparable to the dpp and wg sectors?

123

The implications of this “Regeneration Epiphany” of 1999 are only beginning to emerge [2698]. One minor lesson is the quirkiness of the three legs. Although they share the same basic circuitry, each has special traits. For example, most studies of in situ leg duplication have used an allele (“ts726”) of suppressor of forked that induces apoptosis at high temperature [1501]. When homozygotes are heated during the sensitive period, the frequency of duplications is higher in 2nd legs (ratio for 1st:2nd:3rd legs = 1:32:2) [3442], but the original 1915 study of branched legs (in Morgan’s lab) used an allele of reduplicated [2561] that causes more branching in 1st legs [1875]. Another, more general lesson is that regeneration can follow paths apart from normal development [354] (despite evidence for harmony [548, 1183, 2998, 2999]), so regeneration-based theories must be reexamined. For instance, it is now clear how some fragments can duplicate morphallactically [19, 20, 695] – viz., extant tissue can be repatterned by newly synthesized morphogens. For the same reason, cells in the wound blastema can switch their compartmental afﬁliation [5, 354, 2213, 4223].

The Polar Coordinate Model died in 1999 Because the events described above for leg disc fragments require contact between opposite surfaces, the results afﬁrm the PC Model’s contention that growth is stimulated by such contact. However, in no sense do cells “compute” a “shortest route” to “decide” whether to regenerate vs. duplicate [1472], nor is the growth necessarily “intercalary” sensu stricto. Hence, the PC Model, despite its yeoman’s service as a heuristic aid, seems moribund in light of these ﬁndings. In fairness, a ﬁnal burial should await comparable studies with wing disc fragments, although that corpus of data may also yield to a simple molecular explanation. The wing disc’s peripodial membrane has en-on and -off areas like the columnar side [2072], but its en-on area is larger, so the zone where Hh {dpp and ptc} is shifted anteriorly [2867, 3831, 4404]. Interestingly, the wing disc’s peak of regenerative potency lies squarely in the gap between the A/P lines of the two layers (Fig. 6.1e), and each quadrant (and its 3/4 partner) has one edge in this gap. Although these geometrical clues alone do not solve the puzzle, the problem now seems tractable, and the probes that deciphered leg disc regeneration may soon accomplish the same for the wing disc. Discarding the PC Model also implies dismissing the Angular Values Conjecture (see the start of this chapter). It had tried to reconcile the PC Model with the wedgeshaped expression domains of segment-polarity genes

124

(en, hh, ptc, ci, and wg) by postulating that the latter encode circumferential coordinates [531, 880, 4675]. The ability of some of those genes to cause outgrowths when expressed on the “wrong” side of the disc [231, 1806, 4159, 4848] agreed with the notion that xenotopic confrontations should elicit intercalary growth [354, 532, 1304, 1807, 3862]. However, such growth is apparently not stimulated by confrontations per se because cells can be duped into thinking that they are in the wrong place without causing them to intercalate. Neither the Angular Values Conjecture nor the PC Model satisfactorily explains these ﬁndings. A ﬁtting eulogy for both ideas is provided by the following quote from a paper by Serrano and O’Farrell entitled, “Limb morphogenesis: Connections between patterning and growth.” (See [569, 2455, 3074] for data on which their argument is based; italics are author’s.) Clones expressing a constitutively active form of a Dpp receptor demonstrate that the growth response is an autonomous feature of Dpp signaling. Although cells within these clones over-proliferate in response to the activation of the Dpp signaling pathway, no proliferation -- that is, no intercalary growth -- is induced in the surrounding cells, despite the incongruous juxtapositioning of cell fates at the border of the clone. The unique feature of the transformed cells within these clones is that they have a fate normally associated with a high concentration of Dpp, but lack high Dpp levels. The failure to activate intercalary growth in this circumstance suggests that it is not the juxtapositioning of incongruous cell fates, but rather the juxtapositioning of cells with discordant levels of Dpp that induces this proliferation. Similar data argue that Wg also has a fairly direct role in stimulating intercalary growth. Thus, the discordance that induces intercalary growth appears to be a discontinuity in Dpp and/or Wg morphogen concentrations. . . . The growth factors will diffuse across the junction to stimulate the growth of the cells with low inhibitor levels. [3862]

How Hh, Dpp, and Wg move is not known, nor is their range Hh, Dpp, and Wg signals could theoretically be integrated in many ways to specify fates. For example, in the “Dpp-Wg Growth Potential Model” [3862] cells are supposed to combine Dpp and Wg inputs to compute growth rates. Such a mechanism could be used not only for growth control, but also for patterning. This idea is appealing for the leg disc where Dpp and Wg gradients have opposing slopes but can be forced to coincide, in which case some bizarre phenotypes arise [1812, 2954]. Indeed, both ligands have effects beyond the L2&7 boundary that supposedly separates their domains of inﬂuence (Fig. 5.5a): (1) tkv null clones (whose cells think they are receiving no Dpp) on the V

IMAGINAL DISCS

side of the tarsus produce excess V-type (1/8) bristles and evoke autonomous outgrowths [4277], and (2) dsh null clones (whose cells think they are receiving no Wg) autonomously exhibit excess bristles as far dorsally as rows 3 and 6 [1833]. If, as these data suggest, the Dpp and Wg gradients overlap and are arc shaped, then leg cells could assess their azimuth by computing a ratio of the two inputs. Such “Double-Gradient Models” have historically been appealing because (1) the opposing gradients afford a greater level of precision (especially at low morphogen levels) than single-gradient schemes [91, 1142, 4725] and (2) they let cells ﬁgure out when the ﬁeld reaches ﬁnal size so they can stop growing [2448, 4724]. Such models are hard to evaluate without knowing how far Hh, Dpp, and Wg actually move within the epithelium [641, 783, 1554, 3305, 4275]. Unfortunately, we do not even know how they move [1649, 2448, 3507, 3919, 4275, 4276]. Three chief modes of transit are theoretically possible, not to mention potential routes through the extracellular matrix [277, 2466, 3774, 4275, 4375]: 1. Passive diffusion. Morphogens were originally thought to diffuse randomly through extracellular space [902, 2199, 3087, 3989], and some may in fact do so [674, 2780]. However, free diffusion is unlikely for HhN [1972, 2466, 3432, 4275, 4283], DppC [2004, 3244], or Wg [598, 3561] because they preferentially bind cell-surface proteoglycans and/or lipids [299, 3345, 3854] (cf. glycosylation of Notch, which potentiates its binding of Delta [510]). The epithelial folds in discs should force freely diffusing molecules to skip from crest to crest, whereas the gradients of Hh, Dpp, and Wg deduced genetically imply that the ligands are tracking the surface contours [1910, 4138]. Also, if Hh were diffusing freely, then it is hard to see why changes in cell shape should affect its movement [287]. 2. Active transport via transcytosis. In some systems, proteins can move within the plane of an epithelium, rather than through the extracellular space [1154, 2843]. How they enter and exit cells varies: this “transcytosis” may involve classical endo- or exocytosis or both [1604, 3364, 4562], plus intracellular vesicular transport [811, 4379, 4694] along the same route, perhaps, as recycled receptors [654, 1450] so as to avoid degradation [702, 3217]. Dpp moves via endocytosis in wing discs (dependent on clathrin [1546] and dynamin [1169]), and so may Hh because Ptc (its receptor) resides mainly in lateral membranes and is actively internalized [644, 731, 1022, 1554, 3506]. Hh can traverse ptcnull tissue [277, 754, 755, 4136] but may use an unnatural route to do so. HhN ’s

CHAPTER FIVE. THE LEG DISC

125

cholesterol tail suggests a “Raft Scenario,” where HhN is tethered to “lipid rafts” [1896, 1960, 3595, 3946] that are transferred intact from cell to cell [2297, 4275] in a polarized manner. Alternatively, the sterol-sensing domains in Ptc [255] and Dispatched (a protein that releases HhN from hh-on cells) [572] may be used like hands to grip HhN ’s tail long enough to pass it to the next cell [572, 1975] – a “Tiger-by-the-Tail Scenario.” Anchoring of some kind must be involved because a “tailless” Hh construct escapes control by Ptc and Dispatched to travel 5 times farther than normal and turn on at least one target gene (dpp) throughout the entire A compartment [572]. For Wg, the case is equivocal [1054]. In the embryonic epidermis, Wg moves directionally via a dynamin-dependent (endocytic) route [2900] that is uncoupled from transduction [273, 1055, 1779]. In wing discs, however, Wg is also detected basolaterally outside cells, and this extracellular Wg (signaling ability unknown [1910]) moves independently of dynamin [4138]. Retention (during growth) of Wg-ﬁlled secretory vesicles (in cells whose wg used to be on) may also broaden its range [3363]. 3. Active transport via “cytonemes.” In 1999, the old concept of disc cells as inert “cobblestones” in an epithelial plaza was shattered with the discovery of long ﬁlopodia that extend centripetally from outlying cells to the A/P compartment boundary in wing and leg discs [3507]. Might disc cells be communicating like neurons – via axon-like “railways” [4496]? (Cytonemes seem to use microﬁlaments instead of microtubules.) If so, then morphogens would be taken up at the tips and transported to the cell body [534] (in lipid rafts? [1975, 4275]). This scenario may not be as fanciful as it seems because retrograde transport of morphogens evidently can occur in axons [562, 1933], and the degree of actin polymerization has been shown to affect Hh movement in eye discs [287]. For Dpp, however, this mode of movement is unlikely because Dpp is not detected along cytoneme routes [4265].

Hh molecule. Hh’s range can be doubled by removing its receptor (Ptc) from the cells that it traverses: wherever a ptcnull A clone abuts the A/P line, the dppon stripe is displaced to the anterior side of the clone [277, 754, 755, 4136]. This ability to detach the border zone from the P compartment (cf. Fig. 6.6i) conﬁrms the Hh-Dpp-Wg Model’s logic. The reason that Hh bothers to upregulate ptc transcription [642, 1841, 1982, 2074] must therefore be, at least in part, to reduce its own diffusion range [277, 754, 755, 2074]. 2. Dpp tkv. Dpp can reduce the expression of its receptor (Tkv) [2457], but the level of Dpp that is needed to do so exceeds the physiological range so greatly that this effect is probably irrelevant to normal development [1674]. The virtual complementarity of Tkv and Dpp proﬁles must therefore arise indirectly (i.e., not by a “Dpp tkv” link). Tkv must impede Dpp transit because (1) the slightly higher endogenous level of Tkv in the P compartment is correlated with a steeper response to Dpp [1327], (2) overexpressing Tkv in the dorsal half of the wing pouch shrinks the band where Dpp’s target gene spalt is expressed [4251], and (3) the “wall” of concentrated Tkv outside the wing pouch’s omb domain prevents that domain from expanding when the output of Dpp increases (in dppGal4:UAS-dpp discs) [1674]. 3. Wg Dfz2. The distribution of Wg and its receptor (Dfz2) are roughly complementary in wing discs because Wg represses Dfz2 transcription [595]. Strangely, however, the density of Dfz2 receptors has no effect on Wg movement, possibly due to a low Wg-Dfz2 binding afﬁnity. Indeed, other Wg-binding molecules must import Wg into recipient cells because uptake of Wg into vesicles is seen even in receptor-less (Dfz2 null fz null ) mutants [2984]. Thus, the “Wg Dfz2” link does not illuminate how Wg travels. In contrast to how Wg regulates Dfz2, Wg Dfz3 [3977], although it is unclear to what extent Dfz3 serves as a Wg receptor because Dfz2 null fz null clones in the wing evince no sign of any residual Wg signaling [734].

A further complication is that ligand movement can be constrained by receptor density [674, 775], and all three ligands can regulate the amount of receptor under certain circumstances.

Whether Dpp and Wg travel along curved paths is not known

1. Hh ptc. Hh’s direct effects (as assayed genetically in leg and wing discs) extend over a shorter span (∼10 cells) than those of Dpp or Wg (∼50 cells; cf. Table 6.1), but this limitation is not inherent in the

A priori, diffusion of Hh into the A region would be expected to turn on target genes in zones parallel to the A/P line. The dpp-on “stripe” roughly ﬁts this expectation [487, 3747], but the wg-on domain does not [177, 3317]. Its wedge shape implies that Hh travels farther in the periphery than near the center. As mentioned above, such wedges were once thought to be manifestations

126

IMAGINAL DISCS

Gene expression patterns

Gene circuitry Wide zones

Key:

Key:

ON OFF

gene has LOF effect

activates inhibits e external signal

StPl

e

3 3 3-A 3-A 3-P 3 3 3

dLim1 al PZ63 odz trn HZ84 ap BarH1 PZ22 fas II

3-A 3-P 3 3 3 3-P 3-A 3-P 3 3

Co

Tro

Fem

Tib

T1

T2

T3

T4

T5 * Cl

e Dl e Ser

Dll al

Ser OR Dl

OR ?

dac

Periodic zones

Dll al

al

Dll

Thin zones

BarH1

dac

e

hth esg tsh Dll dac HZ76 ss crol

e

3-P 3-P disco 3-A bib 3-P odd 3-P nub 3 fj 3-P m8 P h P dAP-2 3-P dpn P bab 3-P rn 3-A poxn 3

of angular coordinates [531], but they could be due to the conﬁning of morphogens to circular paths. According to this “Arc Scenario” [358, 1807, 4159], Dpp or Wg molecules stay within the ring (future segment) where they are secreted, although diffusion around the ring would be unimpeded. Different rings could allow different diffusion ranges (e.g., farther at the periphery). Restraints on Wg diffusion have been found at certain folds in the wing disc [595, 3088], but no analogous boundaries are known in the leg disc. An alternative scheme is the “Cloud Scenario” [618, 2456], which assumes random diffusion of Dpp and Wg from their sites of synthesis (Fig. 5.4c). One key difference between these proposals is that in the latter case Dpp and Wg would spread across the center to invade the other’s territory, whereas in the former no “Dpp and Wg” overlap would need to exist.

The debate cannot be settled at present because Wg gradients cannot be resolved ﬁnely enough to assess curvature [595, 4666], and no suitable antibodies exist for Dpp [1546, 1807, 3087, 4251]. Anti-Dpp antibodies used thus far [617, 2457] recognize mainly N-terminal epitopes of the uncleaved precursor rather than C-terminal epitopes of the diffusible ligand [2954, 3244]. GFP-tagged Dpp circumvents this problem [1169, 4265], but the mobility and turnover of the chimeric molecule may be abnormal. Antibodies to phosphorylated Mad reﬂect levels of Dpp transduction [4265], but the only published photo for the leg disc is equivocal [4251]. Hence, the argument has been waged mainly with genetic data. Many genes are known to be expressed in concentric rings within the leg disc (Fig. 5.11) [881]. By itself, this fact could be dismissed as irrelevant to D-V patterning because such domains might reﬂect thresholds

CHAPTER FIVE. THE LEG DISC

127

FIGURE 5.11. Annular domains of gene expression, plotted along the leg’s proximal-distal axis (= peripheral to central in the disc). Genes are grouped based on whether expression is broad, narrow, or iterated, and they are seriated within each group based on proximal limits. Black bars are regions that express the indicated genes (mRNA or reporter) during late 3rd instar (3), early pupal period (P), or adult (A) stage. Degrees of expression are indicated by shades of gray or by graded slopes. All areas are plotted on an adult template, but not all genes are expressed then. The span of stages (e.g., “3-P”) indicates when expression has been seen, but it does not mean that expression is conﬁned to that time. When the claw icon is ﬁlled in, this means that two dots (or one solid spot) are detected where claws arise [1587, 2287], although they could be apodemes [33, 3971], and the “spot” might actually occupy the pretarsus [4401]. See [851, 2287] for histology and [3523] for overview of circuitry. See App. 7 for discussion. Genes whose LOF alleles affect anatomy are in white letters on black background. Wiring at left ( activation; inhibition) shows some genetic interactions (see text or references below at upstream or downstream gene). “External signal” means that the upstream gene emits a signal that affects the downstream gene in nearby cells by diffusion (esg ? dac) or contact (BarH1 ? fasII). Horizontal “T” extensions within the template are conﬁdence limits (not inhibitor icons), although their absence does not imply certainty. Charting is more precise in pupal or adult legs than in discs [999]. Sternopleura (Stpl) comes from the leg disc but is not part of the leg per se (Co, coxa; Tro, trochanter; Fem, femur; Tib, tibia; T1-5, tarsal segments; * = “pretarsus” [2287, 4008]; Cl, claws). Genes (“DO” = details omitted): al (aristaless; DO: Co expression is dorsal only and Tib expression is ventral only) [618, 881] , ap (apterous) [3474] , bab (bric a ` brac) [1516], BarH1 [2287, 3474, 3762], bib (big brain) [999], crol (crooked legs; DO: later spots arise in Tib and Fem) [935], dac (dachshund; DO: dorsal patch outside Tro ring) [4761] , dAP-2 (Drosophila AP-2) [2905], disco (disconnected; DO: squares should actually be bell shaped) [344] , Dl (Delta) [344, 3525] , dLim1 [3474, 4401], Dll (Distal-less) [4761] , dpn (deadpan) [331], esg (escargot) [1573]; fas II ( fasciclin II; DO: the ring’s perimeter is one cell wide) [2287]; fj (four jointed; DO: stripes arise asynchronously) [4852] , h (hairy) [3193] , hth (homothorax) [4761] , m8 (a.k.a. E(spl)) [344], nub (nubbin) [3525] , odd (odd skipped; DO: Fem band may be more proximal) [834] (his Fig. 11d), odz (odd Oz; DO: 2nd leg has more bands in Co and Stpl) [2497], poxn (paired box-neuro) [150], rn (rotund; DO: claws are unstained in some preparations) [881] , Ser (Serrate) [344, 3525] (cf. LOF effects [1944] ), trn (tartan) [721], ss (spineless; DO: expression shifts to Stpl in late 3rd instar; cf. tango) [1166] , tsh (teashirt) [4761] . HZ76, HZ84 (DO: spot in dorsal T5), PZ22, and PZ63 are enhancer-trapped transposons [1574]. Dll ﬁlls the adult tibia in some stained preparations (not shown) [1561] but only the distal half in others [3242, 4761]. N.B.: Most expression domains are dynamic (e.g., see Fig. 5.12b). In late 3rd instar, the hth ring overlaps the dac ring less than the outer Dll ring [4760]. Depending on proximity to the dac-on region, Hth either inhibits or activates tsh (not shown) [4761]. Adult defects imply that dac is needed in T2–3 [2689], BarH1 and BarH2 are needed in T3–5 [2287], and al is needed in T4–5 [618]. In pleiohomeotic LOF ﬂies, the pretarsus looks transformed into a 6th tarsal segment [1500].

in a radial gradient. However, some of these “annulus genes” are ﬁrst transcribed in the D part of their realms, whence the expression spreads laterally and ventrally to close each ring (cf. BarH1 [2287] and dachshund [8, 4575]). In the most extensive screen to date, four “annular” lacZ-tagged transposon inserts were recovered among ∼1200 enhancer-trap lines. Three of the four (HZ84, PZ22, PZ63) showed D-V spreading, and two of these manifested a D-V gradient of expression intensity within the ring [1574]. The following quotes serve not only to document this trend, but also to convey the dynamics of the phenomenon:

est expression remained in the single cell at the presumptive dorsal side of the disc. By the late third instar, the pattern was expressed as a circle in the fused tarsal area. . . . This circular expression formed a gradient with the density of the expression being higher on the dorsoanterior side and lower on the ventroposterior side, as with HZ84. However, this gradient was short-lived. As soon as the larva [pupariated], the expression became uniform and was restricted to the fifth tarsal segment.

HZ84: Expression in the tibial segment expanded gradually to the ventral side of the segment, and just prior to puparium formation, it formed a gradient; the density of expression was higher on the dorsoanterior side, but lower on the ventroposterior side. This gradient was maintained through the early pupal stage, during which it was localized at the distal half of the tibial segment.

One of the three genes associated with these inserts has been cloned and found to contain a zinc ﬁnger motif. Interestingly, another zinc-ﬁnger protein, Klumpfuss (Klu) [4803], also shows D-V spreading [2250].

PZ22: In the early third instar, lacZ was expressed in a single cell at the center of the leg disc. . . . The area of lacZ expression spread from this cell to neighboring cells, but the high-

PZ63: In the leg disc, the expression appeared in the mid third instar at the dorsal side of the coxal segment. . . . The expression extended to the ventral side of the segment, becoming circular and uniform in the late third instar.

Expression in the leg discs starts early during the third larval instar. At this time, the klumpfuss expression domain occupies a wedge-like sector encompassing roughly one third of the circumference of the leg disc. Rings of expressing cells successively become visible underneath a knob-like central

128

structure during the third larval stage. The rings correspond to the anlagen of the leg segments and the order of their appearance reflects the developmental pattern of the leg disc. Around the time of puparium formation, klumpfuss expression seems to be restricted to the distal half of each leg segment in concentric domains, spreading later over the whole leg.

In klu LOF homozygotes, all three leg pairs manifest fusions of the trochanter and femur, as well as of tarsal segments 3–5 [2250]. With severe alleles, the basitarsus is totally missing, but all these losses appear to arise via apoptosis after a normal phase of leg segmentation. Stronger evidence for the Arc Scenario comes from mosaics where parts of the dpp-on or wg-on domains are erased by dpp null or wg null clones. When the source of Dpp or Wg vanishes in a leg segment, the effects (missing or duplicated structures) do not spread beyond the conﬁnes of that segment [1811]. According to the Cloud Scenario, the effects should have spread across segmental annuli. Another fact favoring the Arc Scenario is that V/V and D/D mirror planes in Janus dpp LOF and wg LOF legs coincide with bristle rows [1812] (Fig. 5.5). According to the Cloud Scenario, the planes should have arisen at the cloud perimeter, which would not coincide with bristle rows.

IMAGINAL DISCS

Two of the 4 Hairy stripes are visible in the disc, whereas the other two arise after pupariation. The early stripes – h-onD and h-onV – abut the A/P line and are regulated by Hh, as shown by LOF (h turns off in smo null clones) and GOF (h turns on where ci is misexpressed) studies [1778]. These stripes are also under Dpp and Wg control (h turns off in Mad LOF wg null clones), and separate cis-enhancers at the h locus respond to Dpp vs. Wg. Because dpp and wg are activated by Hh, Hh’s control of h could theoretically be indirect (Hh {Dpp or Wg} h), but the link is actually direct (Hh h) because (1) h stays off in every cell of a smo null clone even when Dpp or Wg is present, and (2) ectopic expression of Dpp or Wg can turn h on only where Ci-155 is also present [1778]. Thus, the circuitry must be combinatorial: the h-onD enhancer responds to {Ci-155 and Mad}, while the h-onV enhancer responds to {Ci-155 and Arm-Pan}. Overall, therefore, the logic is: “{Hh and [Dpp or Wg]} h ac.” One unresolved issue is why a gap arises between the en-on region and the h-onD stripe in pupal legs [1778, 3193] if En Hh and diffusion of Hh h. The gap is 3–4 cells wide and accommodates Ac stripe 4. No such gap should exist if Hh turns h on at the edge of the en-on domain.

Questions remain about the Hh-Dpp-Wg circuitry Hairy links global to local patterning The geometry of the leg disc seems to demand some version of a polar coordinate system, even if the Polar Coordinate Model per se is invalid. In the fate map, the prospective leg segments are nested as concentric rings, and the future bristle rows occupy radial spokes (Fig. 5.1c). It is easy to see how the Arc Scenario could create spokes as thresholds in semicircular morphogen gradients, but the Cloud Scenario’s contour lines would be parabolas that have no obvious relation to spokes (Fig. 5.4c) [1807]. As described in Chapter 3, bristle rows arise within stripes of Achaete-expressing cells. In the tarsus, there are 8 such stripes (Fig. 3.9c). Hairy-expressing cells reside between Ac stripes 4–5 (h-onD ), 1–8 (h-onV ), 6–7 (h-onA ), and 2–3 (h-onP ). This alternation has a causal basis, which is made manifest by disabling hairy. Under these conditions, all the cells that normally express Hairy now express Ac, so 4 broad Ac bands arise (Fig. 3.9f) [3193]. Evidently, Hairy’s duty is to deﬁne Ac stripes by negative regulation (Hairy ac). How Ac is suppressed in the other 4 interstripes (1–2, 3–4, 5–6, and 7–8) remains unknown.

Despite the Hh-Dpp-Wg Model’s success in explaining so many previously enigmatic aspects of leg development and regeneration, additional questions remain: 1. “Yin Yang Paradox”: Why do some cells express supposedly incompatible genes? Given that en ci, it seems odd that cells ahead of the A/P boundary can express both en and ci [350, 4136]. Such overlaps may be trivially due to one gene turning on as its inhibitor turns off in a “fuzzy” transition zone [364, 1561, 2728]. The problem seems more serious for en dpp, although here too the shut-off may simply require a threshold of inhibitor [998]. In mature wing discs, the dppon stripe overlaps the en-on area by 1–2 cells along much of the border but by 3–4 cells at the dorsal edge of the wing pouch [3497] (cf. overlaps of wg-on and enon sectors in leg discs [1472, 4159]). No such overlap is seen in young discs [4228], so the Hh en link may evoke agents (polyhomeotic? [2728, 3512]) that override en dpp at certain places [364]. Much of the zone has a gap (largest in the notal area [3497]) – not an overlap – between the dpp-on and en-on domains [4136, 4479], so thresholds may be region speciﬁc. Intergradations

CHAPTER FIVE. THE LEG DISC

(or lack thereof) may also involve nuances in Ci processing [4478, 4536, 4539] or cross-talk between Hh and Dpp pathways [4251]. 2. “Fragmented Stripe Dilemma”: Why are different parts of the leg’s dpp-on stripe controlled by different enhancers? If Hh turns on dpp in the dAC (cf. Fig. 5.4) by the usual Hh pathway (cf. Fig. 5.7), then wouldn’t it be simplest to use a single cis-enhancer as the “Ci-155 dpp” link [3747]? Indeed, the wing pouch seems to use this sort of strategy [2980]. In contrast, the leg’s stripe is created in pieces – each regulated by a distinct control element [347, 2739, 4056]. We have been fooled before in expecting stripes to be made neatly [49]. Maybe subdividing a stripe into subunits allows each piece’s output to be adjusted to suit each leg segment. The piecemeal strategy makes sense in terms of the Arc Scenario: morphogen output per unit of stripe must increase with distance from the center in order to pump the morphogen around a larger arc. Consistent with this logic, the ﬂy’s distal leg segments are most sensitive to an overall reduction in Dpp signaling [4277], and a centrifugal intensity gradient is seen for the wg-on stripe in grasshopper legs [2069]. Indeed, a preliminary analysis of cis-enhancers at the wg locus shows that the wg-on stripe in ﬂy legs may be subdivided like the dpp-on stripe [2687]. 3. “Leg Stump Riddle:” Why do tarsi tend to be truncated in dpp LOF , but not wg LOF , mutants [528, 529, 1812, 1871]? The rationale in 2 does not help here because the dpp-on and wg-on stripes should behave alike. Nor can the biased loss be ascribed to a need for dpp in only the distal region: dpp must be needed along the entire A/P boundary because a total LOF (via dpp-Gal4:UAS-wg dpp) deletes all D elements and creates legs that are perfectly V/V symmetric from top to bottom [2082]. The answer may lie in how cis-enhancers are arrayed at the dpp locus [347, 2739, 4056]. To wit, the breakpoints of mutants studied thus far might mainly affect tarsal enhancers [834]. Alternatively, a second TGF-β-like gene may act redundantly with dpp proximally. (It would also have to be antagonized by Wg to explain the total V/V phenotype [2082].) Although punt LOF and tkv null somatic clones only affect the distal tibia and tarsus [3329, 4277], repression of punt function for ≥40 h can cause Wg to replace Dpp throughout the entire Dpp sector [4277]. Distal segments are preferentially affected when punt is suppressed for shorter (23 h) periods.

129

Evidently, Punt receives dorsalizing signals along the whole proximal-distal axis (regardless of any redundancy), but the signals may inherently be weaker distally due to the geometry of the disc (cf. 2 above).

Distal-less is necessary and sufficient for distalization As indicated above, Distal-less (Dll) encodes a homeodomain protein [836, 4434] whose expression turns on at the inception of the leg disc [827, 828, 833] and stays on centrally thereafter (Fig. 5.11) [834, 1037, 1544]. LOF alleles cause recessive loss of distal leg structures (hence its name), plus a dominant transformation of distal antenna to distal 2nd leg [1085, 3765, 4212]. Dll function must be necessary for distal cell survival during 3rd instar because (1) chilling a cold-sensitive Dll LOF mutant during this period causes truncations [4212], (2) Dll null clones induced before this time do not survive distal to the coxa [838, 1561], and (3) tarsus-speciﬁc genes (al and bric a` brac) are not expressed in mature Dll LOF discs [618]. LOF alleles can be roughly seriated in terms of the severity of distal loss [839, 4212], but the most sensitive spot is actually the basitarsus (not the tip) [839], and vestiges have gaps that defy ordering [836]. The seriation suggested a proximal-distal (P-D) gradient [839], but Dll expression is uniform in the tibialtarsal region [881, 1037, 1561, 3243], thus dashing any hope that Dll might execute the elusive P-D gradient (e.g., as a “memory” gene) [618]. Besides the disc’s central Dll circle, there is faint expression in the femur and a proximal (∼trochanter) Dll-on ring that arises in 3rd instar in response to other stimuli (including homothorax [4760]). In 1997, Lecuit and Cohen proposed a scheme (here called the “Cloud Scenario”; Fig. 5.4) for P-D patterning [2456] based on how Dpp and Wg affect Dll and dachshund (dac). The dac gene encodes a novel nuclear protein that is expressed in the femur, tibia, and basitarsus [2456], although it is also needed in tarsal segments 2–3 [2689]. In early 3rd instar, the Dll and Dac domains abut one another (solid circle of Dll surrounded by a Dac ring), but by mid-3rd instar they overlap considerably (Fig. 5.12). Because Dll depends on both Dpp and Wg (see above), the overlap of these signals at the disc center might evoke a tip morphogen (“Dpp + Wg = TipM”) whose cone-shaped gradient could specify Dll and Dac at different “T” thresholds (“Dll turns on if TipM > T1 ”; “dac turns on if T2 > TipM > T3 ”). This general idea, the “Distal Organizer Scenario” [488, 618], was implicit

130

IMAGINAL DISCS

Key:

3rd instar Late

Early

D

Dll No overlap

A

a cKey:

Dac

Pleura Coxa Trochanter Femur Tibia T1-5

b

P V

Overlap

d

esg- OFF

Hth

esg- ON

Exd

Esg

dac- ON esg- OFF

?

Dac Exd Hth

esg- ON

e

wild type

T1

T2

T3

T4

T5

Cl

f sple1

ball

g

sockets

ball

V D V

A(4)

B(3)

in Meinhardt’s Boundary Model [2804]. Lecuit and Cohen argued instead that Dpp and Wg diffuse randomly, so Dll and Dac arise in regions of overlap: Dll turns on at high {Dpp and Wg}, while dac turns on at lower {Dpp and Wg}. In that case, Dpp and Wg would act directly. The critical test involved clones whose Dpp (tkv GOF ) or Wg (sgg LOF ) transduction pathway is activated in “alien” territory (V or D region, respectively). Such

C(4)

D(3)

E(2)

F(4)

clones (unlike clones that emit Dpp or Wg) fail to induce Dll or Dac in surrounding cells [2456]. Ergo, the Distal Organizer scheme must be wrong. (TipM should have seeped out and induced Dll and Dac.) TipM could still be an instructive signal if cells also need permissive levels of Dpp and Wg to turn on Dll or dac (e.g., “Dll turns on only if TipM > T1 , Dpp > TDPP , and Wg > TWG ”) – a condition met inside, but not outside, the clone.

CHAPTER FIVE. THE LEG DISC

131

FIGURE 5.12. Proximal-distal axis of the leg and how it is subdivided into discrete domains.

a. Blank templates for charting zones of gene expression: disc (left) and derivatives (right) consisting of pleura (body wall) and the leg proper (9 segments; cf. Figs. 5.1 and 5.11). Abbreviations: Dac (Dachshund), Dll (Distal-less), Esg (Escargot), Exd (Extradenticle), Hth (Homothorax). b. Areas where Dac and Dll are expressed during 3rd instar. The Dll circle initially abuts the Dac ring but later expands to overlap it. (Note also the emergence of an outer ring.) An opposite situation occurs more distally (not shown) where an Aristaless circle and a Bar ring ﬁrst overlap and later abut [2287]. c. Areas where Dac and Esg are expressed (left) and intercalation of a dac-on zone wherever esg-on and esg-off cells abut (right). Dac and Esg zones normally overlap slightly (omitted at right). Two clones (circles) are cartooned. When an esg null clone (above) is induced in esg-on territory (where Esg and Dac overlap [1573]), its peripheral cells make Dac. When an esg GOF clone (below) arises in esg-off territory, Dac is expressed by cells around the clone. Evidently, esg-on cells emit a signal that causes nearby esg-off cells to turn dac on (Esg ? dac). An analogous induction (Bar contact? fasciclin II) occurs more distally (not shown) [2287]. d. Proximal vs. distal domains of the leg disc. In the proximal area (black; dorsal region exaggerated), hth and esg are expressed (Hth esg), and Hth enables Exd to enter the nucleus (Hth Exdnuc ) [9, 130, 677, 2673, 3589]. In the distal area (white), hth is inhibited jointly by Dpp and Wg so Exd cannot enter the nucleus [8]. The inhibition involves high doses of Dpp and Wg but apparently is not mediated by Dac or Dll [4760], so its route is unclear. The likely agent until early 3rd instar is Antennapedia (Antp hth), but Antp fades from the endknob by mid-3rd instar [677]. e, f. Extra tarsal joints caused by mutations in “planar polarity” genes. “T1–T5” are tarsal segments, and “Cl” denotes claws (dorsal is at top, ventral at bottom). e. Tarsus of wild-type 2nd leg (bristles omitted) showing normal ball-and-socket joints (solid black). Within each joint, the ball belongs to the distal segment, whereas the socket belongs to the proximal one. Pivoting of balls inside sockets lets the tarsus curl ventrally when an apodeme (not shown) is pulled by tibial muscles (like a marionette) [236, 2857, 3421]. The joints arise dorsally [1812] and constrain movement to the D-V plane (like an elbow), although the details of their morphogenesis are unclear [4009]. Note unequal lengths of T1–T5 and ﬂared tip of T4/T5 socket. f. 2nd-leg tarsus from a ﬂy homozygous for the spiny legs 1 (sple 1 ) mutation (a.k.a. pk sple1 ) at the prickle locus [1641, 1795, 1810, 2884]. In each segment (except T5, which is immune), there is an extra joint. The extra joint resides near each segment’s midpoint (except in T1, where the joint lies distally and only makes a partial intersegmental membrane). All extra joints are upside down (balls proximal to sockets; cf. the embryo’s “segment-polarity” phenotypes [3151]). The extra joint in T4 has a ﬂared socket like the T4/T5 joint, so joints are probably copies of their segment’s distal (vs. proximal) end. Mutations in other planar-polarity genes (e.g., fz or dsh) cause milder phenotypes [1810, 4716]. One clue to the etiology is that ommatidia, like joints, are ensembles controlled by the same polarity circuit. In that case, the whole proneural group is oriented by a few key cells (cf. Fig. 7.5) [4366], so maybe the same is true for joints. g. “Fickle Sensilla Mystery” associated with the double-joint syndrome (cf. Fickle Bristle Mystery; Fig. 7.8). Hourglass shapes are full-surface views of the distal half of T3 from legs of various sple 1 individuals. Dorsal (D) and ventral (V) midlines are marked. Black dots are sensilla campaniformia (stretch receptors [4342]). Types of sensillar patterns in a sample of sple 1 legs are shown, with numbers of legs per class (A-F) in parentheses (total = 20). In wild-type ﬂies, two sensilla always arise at the distal end of T3 (as in A) and T1 (not shown) [3705]. In the mutants, sensilla must only arise near the normal joints (not near the ectopic ones) because otherwise there would be more than two sensilla. However, they must then be able to move to the extra joint. Normal and extra joints seem to “attract” sensilla equally (cases of A ≈ B, and cases of C ≈ D), with occasional cases (E or F) where a sensillum cannot seem to decide and hence has been frozen in midsegment. Panel b is adapted from [2456, 4497, 4575], c depicts data from [1573], d is based on [8], e and f are traced from [1810] (although the sple 1 tarsus is actually ∼10% shorter than the wild-type one), and g depicts data from [1810]. See [883, 1341] for an intercalation scheme.

Additional experiments conﬁrmed that Dpp and Wg turn on Dll and dac at different thresholds, consistent with the Cloud Scenario. The Arc Scenario seems defunct because it does not explain how Dpp and Wg can achieve stepped plateaus of combined activity coincident with the Dll and Dac domains. However, as mentioned above, each part of the dpp-on and wg-on sectors seems to be governed by separate cis-enhancers [347, 2687, 2739], and thus the central output of Dpp and Wg could be higher than in the periphery. Admittedly, the “Dpp and Wg” proﬁle would be uneven (low near D and V

midlines and high at A and P midlines), but it might still exceed a basal threshold for Dll activation throughout the central area. Thus, the Arc Scenario is still viable, although now laden with ad hoc assumptions. Given the dependence of Dll and Dac on Dpp and Wg, it came as a surprise when Dll and dac were found to stay on after the Wg (dsh null clones) or Dpp (tkv null or Mad LOF clones) pathways are shut off after 2nd instar [883, 2456]. The inference was that Dpp and Wg are needed to turn on Dll and dac but not to maintain the on states. Lecuit and Cohen thought that Dll’s on state

132

might be perpetuated by cell lineage, but subsequent studies showed that the Dll-on state is not heritable [618, 1561, 4575]. The reason for Dll’s and dac’s endurance remains unclear. Interpretation of these results is complicated by some quirks of the circuitry. First, overexpressing Dll in its own area – a GOF manipulation – causes a LOF phenotype (leg truncation) [1561]. This effect implies downregulation of the endogenous gene via “Dll Dll” autoregulation. More important, ectopic clones of Dllexpressing cells induce leg duplications with expression of dpp and wg inside the clone [1561]. This “Dll {dpp and wg}” link seems to violate the rules of normal development (dpp and wg are only on in subregions of the Dll circle), although it may only operate at Dll levels above the physiologic range. Evidently, Dll is not only necessary for distalization, but also sufﬁcient to induce it. Dll also shows some biases along the D-V axis: (1) Dll function is essential in the ventral, but not dorsal, femur [618]; and (2) Dll LOF interacts strongly with wg LOF but weakly with dpp LOF [1812].

Proximal and distal cells have different affinities Dll null clones induced during 3rd instar grow (albeit slowly) in segments distal to the coxa and form vesicles that detach from surrounding Dll + tissue, except in the femur and upper tibia (where they integrate normally) [618, 1561]. The bristles in the vesicles differ from those in the overlying epidermis, suggesting a switch in regional identity [1561]. Evidently, Dll-on and -off cells have different afﬁnities [3242, 4760]. P-D afﬁnity differences were discovered long ago in moth wings by transplanting epidermis along this axis [3047, 3049, 3050]. Dll does not affect afﬁnities in ﬂy wings [618], where Wg (or another Wnt) instead must preside because fz null Dfz2 null clones in the hinge are round with smooth edges [734]. Further evidence for P-D cell sorting comes from studies with other transcription factors. From the inception of the leg disc when Dll appears distally [1572], the proximal cells express homothorax (hth, homeobox class) [2361, 3226, 3589] and its downstream target escargot (esg, zinc-ﬁnger class) [1542, 1573, 1765]. When hth GOF or esg GOF clones are induced in distal territory (where both genes are normally off), the clones detach from the epidermis and form vesicles [1573, 4760]. What role, if any, does this immiscibility play in the wild-type leg disc? One idea is that it prevents cells from crossing a boundary near the future trochanter [4760]. This line appears to be a legacy of how the arthropod

IMAGINAL DISCS

leg evolved [1544, 2937]. Even if this exclusionary rule is true, however, cells must still be able to change their afﬁnity states (cf. the Cabaret Metaphor; Ch. 4) because the P-D axis has no lineage restriction at the trochanter or elsewhere [544, 618, 1561, 4076, 4575] (cf. a similar situation in the notum [614]). Consistent with this reasoning, cells that are permanently unable to turn on Dll (Dll null clones) apparently fail to cross the P/D line [4760]. Like the P/D line, the D/V line that divides the disc into dpp- and wg-dependent sectors (Fig. 5.5a) is also maintained without lineage barriers. Might it also separate immiscible (D vs. V) populations [4760]? Indeed it may, because sgg null clones (whose Wg pathway is permanently on) often form vesicles when they are induced in 1st instar outside the V region (cf. similar behavior in the wing and notum [3603, 3956]). All sggnull clones in dorsal positions cause ectopic growths in which the mutant tissue rounds up and appears to minimize contact with non-mutant cells. This correlates with the adult phenotypes of dorsal sggnull clones where the mutant cells are often displaced distally in ectopic outgrowths. [1833] sggnull clones in lateral positions cause simple outgrowths of leg. The outgrowths consist entirely of genetically marked sggnull mutant cells, which adopt ventral-most identity. . . . Since the fates of the mutant cells are inappropriate for their position in the leg, the clones are apparently unable to integrate into the normal pattern and the mutant cells are extruded from the leg as outgrowths. . . . We have also observed clones of sggnull mutant cells, which segregate internally to form vesicles. The vesicles are composed of mutant cells, which produce genetically marked ventral bristles projecting into the lumen. Since the vesicles are often detached from the leg, it is not possible to know where these clones of ventral cells originate; however, it is possible that they form in lateral positions and segregate internally. [1039]

Vesicles are also seen with dsh null clones [4278] so the effect is not gene speciﬁc, although it may only be manifest by the Wg (vs. Dpp) pathway along the D-V axis. The D/V immiscibility must eventually wane because sgg null clones induced in 2nd instar integrate normally at virtually any point around the disc circumference [4666]. Long before the P-D and D-V axes were suspected of having afﬁnity subtypes, such differences were implicated along the A-P axis [2441, 2928]. Because most investigations of A-P afﬁnities have been pursued with the wing disc, that corpus of work is discussed in Ch. 6.

Dachshund is induced at the Homothorax/ Distal-less interface Despite the absence of true P/D compartmentalization, the segregation of (albeit transient) proximal and distal

CHAPTER FIVE. THE LEG DISC

cell types could allow other genes to be turned on at the interface, analogous to how Hh turns on dpp and wg at the A/P interface. Indeed, such signaling does seem to occur. Both hth GOF and esg GOF distal clones elicit expression of dac in nearby wild-type cells (Fig. 5.12) and disturb their polarity [1573, 2825]. These nonautonomous effects are probably mediated by a diffusible signal, which – given the polarity defects – could be a Wnt: Hth Esg Wnt? Dac. This inference is hard to reconcile with Lecuit and Cohen’s surmise that dac responds autonomously to Dpp and Wg [2456]. Conceivably, as argued above, Dpp and Wg may only act permissively (albeit in a threshold-dependent manner). This process whereby a third state (dac-on) emerges at the interface between two others offers a way of understanding intercalation [1341] without the need to invoke coordinates per se (e.g., the Polar Coordinate Model [1303]). Similar logic might explain intercalary regeneration in hemimetabolous insect legs because polarity reversals arise there when a donor leg is severed proximally and grafted to the stump of a leg amputated more distally (e.g., see [3878]). Certainly, the results help demystify the old “Ends-Before-Middle Riddle” of why young discs fail to make mid-level P-D structures when they undergo premature metamorphosis [3809]. Namely, intermediate identities would emerge stepwise as the pattern is elaborated during growth [1573]. In fact, Dac only begins to be expressed at the interface between hth-on (outer) and Dll-on (inner) regions at the end of the 2nd instar [8, 2287, 4575, 4760]. Presumably, the “Esg Dac” link is only activated at that time (i.e., a temporal trigger). Alternatively, a critical mass of signaling cells may be needed to overcome a response threshold (i.e., a scalar trigger). Other peculiarities of the leg’s P-D axis have been uncovered over the years. Within each leg segment, for example, distal bristle SOPs tend to divide before proximal ones [1598, 1803, 1808, 3142]. Mitoses, in general, orient along the P-D axis during 3rd instar [544] – a trend that explains the narrow shapes of wild-type clones [3441, 4344], which can be 100 cells long but only 3 cells in width [544]. In conjunction with a higher rate of distal growth (spurred by more Dpp?), this radial pattern of growth may also explain why the epithelium buckles into concentric folds during this period.

Homothorax and Extradenticle govern the proximal disc region As mentioned earlier, the proximal part of the leg disc (pleura to trochanter) expresses hth, while the distal

133

part (trochanter to tip) does not [677, 1573, 3589]. These areas also roughly delimit the action of extradenticle (exd, it is also needed in the proximal femur [1544]), another homeobox gene [1248, 3526]. Although exd is transcribed uniformly in the disc [1248, 3527], Exd’s subcellular location differs proximally (nuclear) vs. distally (cytoplasmic) [130, 2673] as does its amount [1543, 3226]. Exd’s distribution is dictated by Hth [9, 2045, 3716]: Exd localizes to the nucleus (“Exdnuc ”) wherever Hth is ectopically expressed but localizes to the cytoplasm (“Exdcyt ”) when Hth is removed [8, 302, 677, 3226, 3589]. In turn, Exd sustains Hth: Hth vanishes in exd null clones, although hth is still transcribed, so Exd may block Hth degradation [8, 2045]. These links make sense because Hth binds Exd [9, 2045, 3589, 3716] and these heterodimers act as transcription factors to activate downstream genes [2045, 3716]. One of those targets is esg : esg is turned on in distal hth GOF clones [1573]. The ﬁrst three links below would operate proximally, while the fourth would evoke Dac (via a paracrine signal) in nearby distal cells. Link 1: Link 2: Link 3: Link 4:

Hth Exdnuc . Exd Hth. {Hth and Exdnuc } esg. Esg Dac (3rd instar only).

Thus, the leg disc has a proximal ring where HthExdnuc heterodimers govern one set of target genes and a distal core where Dac and Dll might orchestrate a different set (Fig. 5.12d). Dac and Dll, in turn, are controlled by Dpp and Wg – the ultimate sovereigns of the distal province (although Dpp [4277] and Wg [177, 519, 1812, 3317, 4278] are also needed proximally). Indeed, some targets of Dpp alone (omb) or Wg alone (H15) are expressed in sectors that abut the hth-on ring, even though the dpp-on and wg-on sectors extend into that ring (Fig. 5.8) [8,1542, 1544]. Could Hth-Exdnuc be overruling the commands of Dpp and Wg? Yes, because (1) expression in the omb or H15 sectors vanishes wherever hth GOF clones overlap them, (2) omb and H15 are derepressed in proximal hth LOF clones (in D or V regions, respectively) [8], and (3) omb is turned on by proximal exd LOF D clones and off by distal exd GOF D clones [1542]. Hth’s veto of Dpp’s (and Wg’s?) authority is not mediated by esg because esg GOF clones do not turn off omb distally [1573]. Link 5: Link 6:

Hth Hth

{Dpp {Wg

omb}. H15}.

An initial report suggested that Dac and Dll are snuffed out in hth GOF clones [8] (excess Exd alone has no effect [1542, 1544]), in which case Hth would also block

134

IMAGINAL DISCS

Dpp and Wg’s joint targets. However, more recent data refutes the “Hth Dll” link and possibly the “Hth dac” as well [4760]. Rather, Dll and dac may be off proximally simply because peripheral Dpp and Wg signals are too weak to prod them into an on state [2456]. Based on the earlier work, a “Mutual P-D Antagonism Model” was proposed wherein (1) distal states are supposedly suppressed in the proximal area by the disputed “Hth Dll” and “Hth dac” links, and (2) proximal states are supposedly inhibited distally by the reciprocal “Dll hth” and “Dac hth” links [8, 1542]. However, the latter two links also appear to be false [4760] due at least in part to illusions involving the folds in the disc epithelium. One consistent ﬁnding is that proximal cells turn off hth [8, 4760] and hence replace Exdnuc with Exdcyt [1542] whenever they “think” they are receiving high doses of both Dpp and Wg (viz., V clones that are tkv GOF or D clones that are wg GOF or arm GOF ). Thus, the only conﬁrmed inhibitors of proximal states in the distal region are Dpp and Wg, although it is unclear how these signals cooperate and whether they use proxies (excluding Dac and Dll). Link 7:

{Dpp and Wg}

hth.

Given the reciprocity of Links 5 and 6 on the one hand (i.e., Hth’s damping of Dpp and Wg transduction) and Link 7 on the other hand (i.e., Dpp and Wg’s damping of Hth), the basic premise of the Mutual Antagonism Model may still be correct, and the immiscibility described above could make sense as follows. If hthon cells are less adhesive than hth-off cells, then they would automatically sort to the periphery [4071--4074] and that outer ring would be desensitized to Dpp and Wg [4760]. The inner cells would then be left to form a central “plateau” where the {Dpp and Wg} combination turns on {Dll and Dac} in neat circles despite its saddle shape. The humps of the saddle could be effectively ﬂattened during transduction by damping factors such as Dad and Nkd. Do Hth and Exd control proximal vs. distal cell identities per se? Dramatic evidence that they do so comes from proximal shifts in the identities of distal cells when they are forced to express hth: tarsal segment T4 makes basitarsal (T1) sex comb teeth in ap-Gal4:UAS-hth ﬂies, and hth GOF clones make femoral bristles in the tibia [2825] (cf. large bractless bristles in distal hth GOF clones [1573]). These are the kinds of shifts expected if Hth mufﬂes a cell’s ability to “hear” (or act upon) Dpp and Wg signals [4760]. Moreover, the mufﬂer would conﬁne Dpp’s mitogenic effects to the distal region and thereby fa-

cilitate appendage outgrowth from the body wall [2937]. Proximal exd LOF clones cause reciprocal shifts: they tend to make distal-type (small bracted) bristles (e.g., tibial bristles in the trochanter) [1543, 1544]. No P-D shifts have been reported for proximal hth LOF (or esg LOF ) clones or distal exd GOF clones, while distal esg GOF clones only make smooth cuticle [1573]. Curiously, exd LOF discs that fail to form coxa or trochanter still express the proximal marker genes teashirt and odd skipped during 3rd instar [3527]. Like esg, teashirt (tsh) encodes a zinc-ﬁnger protein [66, 1197] that could control the transcription of identity-implementing genes (a.k.a. “realizator” genes [1358]), but distal tsh GOF clones do not alter P-D identities [1175].

Fasciclin II is induced at the BarH1/Aristaless interface Subtler shifts in P-D identity are seen when BarH1 is overexpressed. The “Bar” gene – made famous by Sturtevant’s studies of unequal crossing over [4179, 4186] – turned out to be two redundant homeobox genes BarH1 and BarH2 [1842]. During 3rd instar, both genes are strongly expressed in tarsal segment T5 and weakly in T4 [2287]. When BarH1 is overexpressed in T4 (by an ap-Gal4 driver), T4 bristles now resemble those of T5. In contrast, LOF clones homozygous for a deﬁciency of the Bar locus show a transformation to T3 [2287]. The implication is that the Bar-off “ground state” dictates T3, medium amounts of Bar dictate T4, and a high level dictates T5 [2287]. The leg disc’s T5 ring surrounds densely packed “pretarsal” cells that express aristaless [851, 2287]. The BarH1 and Al domains initially overlap, but later (by mid-3rd instar) they abut along a sharp boundary. This line seems (based on LOF-GOF data) to be maintained by mutual repression: “BarH1 al” and Al BarH1” [2287, 4401]. Both domains are subsets of the Dll territory, and both genes are permissively kept on by Dll: “Dll BarH1” [2287] and “Dll al” [618]. Moreover, BarH1 helps ban Dac from part of the Dll domain because BarH1 dac [2287]. In late 3rd instar, the outermost pretarsal cells (those that touch BarH1-expressing cells) begin expressing Fasciclin II (Fas II) [2287], a homophilic adhesion molecule [1620, 4758]. Might BarH1 be inducing Fas II? If so, then the induction would likely involve a BarH1 target gene that encodes a ligand, with the ligand ﬁtting a receptor on al-on cells: BarH1 Ligand? Receptor? Fas II. Support for some sort of induction comes from both LOF data (BarH1 null clones erase Fas II from its

CHAPTER FIVE. THE LEG DISC

normal domain) and GOF data (Gal4:UAS-driven expression of BarH1 in a stripe within the Al circle elicits Fas II in two ﬂanking lines of cells) [2287]. Because the responding cells (endogenous or ectopic) always occupy single ﬁles, the induction mechanism is probably mediated by direct cell contact (i.e., a cell-surface ligand), rather than by diffusion (cf. the “Esg Dac” induction). Fas II-expressing “border” cells have a unique rectangular shape apically [851, 2287, 2408]. Conceivably, the homophilic Fas II molecules might be “zippering” these cells into a ﬁle by maximizing contact. These tensile forces might also help the Fas II ﬁle keep its hoop shape, although the hoop is probably not stressed by growth because (1) it arises so late that there is little further proliferation, and (2) inner cells are mitotically quiescent [1599, 4666]. Al may play an executive role in cell adhesion or polarity [4350], as may dLim1 – a LIM-homeodomain protein that is co-expressed with Al [4401].

BarH1 and Bric a` brac affect P-D identity, joints, and folds As discussed above, BarH1 seems to specify T4 and T5 states relative to T3 by its level (high in T5, lower in T4, absent from T3) [2287]. A similar role seems to be played by the gene bric a` brac (bab, named after ovary defects [3722]), although bab’s “ground state” is T1. Bab (a BTB-domain protein) localizes to the nucleus and may inﬂuence target genes [1516]. Bab is expressed strongly in T4 and T3, less in T2, and minimally in T1. Strong LOF alleles of bab transform T2, T3, and T4 to resemble T1 [1516] – an effect that is dramatized by T1-like sex combs on T2, T3, and T4 of the male foreleg. Weaker alleles only affect T2. The sensitive period occurs shortly after pupariation [1516]. A T1 ground state makes sense because so many Drosophila species have combs not only on T1, but also on distal segments [376, 1361, 3752, 4095]. Flies can be coaxed to make combs on segments distal to T1 by the following agents: 1. Treatments that cause cell death: X-irradiation [4343, 4349], nitrogen mustard [4336], or surgery [4143]. 2. Apoptosis-inducing mutations in suppressor of forked [3441, 3442]. 3. Growth-perturbing mutations in comb gap [962], dachs [4348, 4515], four jointed [4348, 4515], hyperplastic discs [2685, 2709], l(1)ts504 [3964], or Notch [3525]. 4. Homeotic mutations in cramped [3752, 4793], dachshund [3752], Enhancer of zeste [3752], extra sex combs [4095], multi sex combs [3721, 3755], pleiohomeotic [1500],

135

poils aux pattes (whose product is part of the Mediator complex) [412], Polycomb [645, 1024], proboscipedia [3333], sex comb distal [411, 3752], spineless [4095, 4515], and other genes [2561]. Evidently, T2, T3, and T4 of the male foreleg are predisposed to make sex combs (i.e., have a “prepattern”), but only T1 normally does so because bab and other genes block the realization of this potential in the T2–T4 region (i.e., suppress “competence”) [4095]. This surmise is supported by the fact that several of the above genes belong to the Polycomb Group of repressors (cf. Ch. 8) [3752]. Aside from their different ground states (T1 vs. T3), Bab also differs from BarH1 in that it is expressed in a gradient within its respective segments (Fig. 5.11). Iterated gradients of positional information have long been suspected for insect leg segments based on (1) regeneration outcomes seen after grafting legs of cockroaches or other hemimetabolous insects, and (2) polarity disturbances when ﬂy leg segments partially fuse in various mutants [386, 3427]. However, Bab probably is not serving this function because no intrasegmental displacements of sex combs (away from its normal distal site) were reported for bab LOF mutants. (They should have occurred if Bab were specifying P-D position within each segment.) Bab and BarH1 both must govern the T4/T5 joint (where their most intense zones abut) because T4 often fuses with T5 in bab LOF mutants, Bar null clones, and BarH1 GOF (ap-Gal4) tarsi [1516, 2287]. The ability of these genes to induce a joint invagination may be related to their ability to induce folds in 3rd-instar leg discs at their domain boundaries [1516, 2287]. LOF alleles of bab also affect the T2/T3 and T3/T4 joints, but the effects are milder than for T4/T5.

Leg segmentation requires Notch signaling Mutations in various Notch pathway genes cause fusions of tarsal segments [881]: Notch [344, 999, 3525, 3891], Delta [344, 999, 3277, 4466, 4467], deltex [1570], fringe [999, 3525], kuzbanian [4025], Serrate [344, 4029], strawberry notch [895], Suppressor of Hairless [999], etc. [2195, 4467]. Many of these same mutations also prevent the detachment of the distal femur from the proximal tibia [999, 3525] (cf. groucho LOF [998]), which normally occurs during the early pupal period [1311]. The involvement of the Notch pathway in leg segmentation was analyzed in 1998–99 [344, 999, 3525]. By turning on the pathway (via Notch GOF , Delta GOF , or Serrate GOF ) in unusual locations, extra joints could be

136

induced [344, 999, 3525] (cf. Bar null [2287]), implying that the pathway is sufﬁcient as well as necessary for creating joints. For example, when Serrate (a ligand) was artiﬁcially expressed in the middle of the tibia (using a Gal4 driver), two new joints arose there – one at either end of the ectopic Serrate domain [344] – implying that joints are made at Serrate on/off boundaries. Normally Serrate is only expressed at the distal end of each leg segment (Fig. 5.11), so this experimental result begs the question: why doesn’t a joint normally arise on the proximal side of each Serrate band as well as on the distal side? There is no obvious answer because all the needed components of the transduction pathway are present on both sides. One clue to this mystery is a “double-joint” syndrome where an extra joint does indeed arise on the proximal side of each Serrate band in T1–T4 [344, 1810] in addition to the normal joint, and each extra joint is upsidedown (Fig. 5.12f ). The syndrome is caused by LOF mutations in frizzled (fz), which encodes a receptor for Wg and maybe other Wnts, and dishevelled (dsh), which encodes a transducer for Wg and maybe other Wnts. Conceivably, a Wnt signal normally polarizes cells so that they can tell whether they are on the “right” or “wrong” side of the Serrate band. When that signal fails, cells on both sides think they are on the right side and hence make mirror-symmetric joints [344]. This idea is at least plausible because cross-talk between the Notch and Wingless pathways occurs in the wing disc [459, 886, 2718, 2839, 3690], and Dsh can bind Notch [151, 356]. A separate pathway involving the LIM domain protein Prickle also suppresses extra joints [1641, 1810], but the nature of that circuitry remains to be elucidated. The annular expression domains categorized in Figure 5.11 are reminiscent of the hierarchy of embryonic segmentation genes (Fig. 4.2) [1341, 1988] – the “wide zone” class corresponding to gap genes and the “periodic zone” class to segment-polarity genes [3523]. Body and leg segmentation are similar processes of “slicing” a cylinder into rings, but does this analogy have any real mean-

IMAGINAL DISCS

ing? Could the leg be a scaled-down version of the body [2868]? Unlike body segmentation, leg segmentation does

not involve cell-lineage restrictions [2852]: clones can cross segment boundaries virtually whenever and wherever they are induced [544]. Nevertheless, the doublejoint syndrome may have an etiology related to segment-polarity phenotypes because dsh LOF causes both traits [344, 1810]. Two pair-rule genes (hairy and odd skipped) are in the leg’s periodic class and another (odd Oz) has a single ring, but there is no evidence of any two-segment periodicity in the anatomy or sequence of disc folds [834, 1516, 2287]. As a rule, wide-zone genes are expressed earlier than periodic-zone genes (echoing the embryo sequence). The Notch pathway is not involved in body segmentation in ﬂies [3695, 4601] but may have been used ancestrally [1009, 3525]. A ﬁnal answer regarding bodyleg homology will be impossible until we know much more about how both body and leg circuitry evolved [1807, 1988, 2069, 2868, 3122, 4230]. The analogy at least has heuristic value. For example, if combinatorial inputs from the wide-zone class dictate periodic-gene expression patterns [3525], then the latter’s cis-enhancers should bind trans-effectors from the former. The fact that several wide- and thin-zone genes encode putative transcription factors (e.g., homeobox genes hth, Dll, al, BarH1, and zinc-ﬁnger genes esg and tsh) is compatible with this scheme. The periodic-zone gene disco could mediate joint formation: it encodes a zinc-ﬁnger protein that controls intercellular adhesion [1791, 4079], perhaps the kind of adhesion needed for joint invagination [1311, 2468]. Regardless of its circuitry, this system is worth studying for its robustness [4515], especially its ability to reliably make exactly 9 segments. An equivalent robustness is evident in the wing disc, where two symmetric surfaces (each with 5 longitudinal and 2 cross veins) are created separately and then plied together with virtually no mismatching of dorsal vs. ventral elements. Accuracy is of the utmost importance in that case because a defective airfoil is a debilitating handicap for a ﬂy.

CHAPTER SIX

The Wing Disc

The A-P axis is governed by Hh and Dpp but not by Wg Like the leg disc, the wing disc uses Hedgehog to set up a border zone just ahead of the A/P compartment boundary [231, 4228] (cf. Fig. 5.7), but its zone emits only one long-range morphogen – namely, Dpp [231, 354, 2448]. Wingless is irrelevant for the wing’s A-P axis and instead functions along its D-V axis [4849]. Both morphogens are essential: wings fail to develop when the disc is deprived of either Dpp [529, 3438, 4032, 4848] or Wg [884, 4683]. Topologically, the wing is like a squashed leg (Fig. 6.1). Its D and V faces are apposed, and its veins run along its length like the leg’s bristle rows. However, while the prospective bristle rows converge centrally in the leg disc (cf. Fig. 5.1), the primordia of veins 2–5 are parallel to one another and intersect a perpendicular line (the future margin). Thus, it is unclear whether the wing has a true “tip” like the leg [4682]. Certainly, the expression of Dll in a band along the wing margin (Fig. 6.2) differs from the circle of Dll in the leg disc (cf. Fig. 5.4) [2254]. The stripe where dpp is expressed in a mature disc is ∼5 cells wide [754, 2739, 4188, 4251, 4479]. That width is the net result of (1) activation of dpp by Hh (Hh dpp) in a larger zone (8–10 cells wide; cf. Fig. 5.7) and (2) repression of dpp by En (En dpp) in the rear 1/3 (3–4 cells) of that zone [350, 2728, 2992, 4136] in the same way that En represses dpp in the P compartment throughout larval life [1647, 3747, 4229] (but see Fig. 6.3). Suppression of dpp in the border zone is only seen in late 3rd instar [350, 364, 4228] when a “Hh en” link turns en on in A cells that receive Hh [78, 642, 3818]. As was argued in Ch. 5 (the Minotaur Scenario), this new link could join the old “en hh” link [1647, 1659, 4227, 4848] to spark a chain

en Hh”), but is prevented from doreaction (“Hh ing so in the leg disc by Dpp or Wg. In the wing disc (Fig. 6.4a), the “circuit breaker” is Dpp alone (Dpp {Hh en}). Overall, therefore, the logic consists of the 5 links listed below, but until recently it was unclear how Hh-responsive cells decide whether to activate (Link 1) or repress (Links 2 and 3) dpp. Link 1: Link 2: Link 3: Link 4: Link 5:

Hh dpp. Hh en (late 3rd instar only). En dpp. en hh. Dpp {Hh en}.

In 1997, Strigini and Cohen showed that this circuitry works not just in the border zone but anywhere in the A compartment [4136]. When they induced hhGOF clones in the A region, en turned on in every clonal cell and in nearby wild-type cells that got Hh by diffusion (Link 2). At a still greater radius (rdpp > ren > rhh) were dpp-on cells (Link 1). Unlike the hh-on and en-on “biscuits,” however, the dpp-on domain was a “doughnut” with a dpp-off hole in the middle where Link 3 evidently overrules Link 1. These clones could presumably have pushed their en-on state out farther into the A region by alternating Links 2 and 4 (“Hh en Hh”), but they did not, evidently because the doughnut acts like a cage to block them (Link 5) [2082]. Given this policing ability of Dpp, how does the en-on state normally creep (albeit weakly [350]) from the P into the A region at all? Maybe the intrusion reﬂects a balance of forces, with Link 2 barely subduing Link 5. Strangely, Zecca et al. had obtained different results from similar experiments in 1995 [4848]. They saw dpp 137

fate map

PS

1

stalk

NP

ra eu ge pl in h

a

100 µm

IMAGINAL DISCS

43 cells

138

c

A V

D

*

P

SA

DC p

a

*A

P

top view

3

DV 4

ation agin ev

hinge

2

A D P V crosssection D N V N

5 PA PS

d *

SC

a p

SA

NP

hinge

b

1 2 3

e

A P

4 5

D

A PM P * CE

A P

PA

heminotum & wing fragment duplicates Key

f

Peak of regenerative potency

fragment regenerates

α

γ β

doughnuts around enGOF A clones, but their hhGOF A clones expressed dpp inside as well as outside each clone (i.e., biscuits vs. doughnuts), thus disobeying Link 3. This malfunction in Link 3 is traceable to a glitch in Link 2 because their hh transgene did not turn on en as Strigini and Cohen’s transgene did. Why not? Apparently, the constitutive promoter they used (Tubulinα1 vs. Actin5C) was too weak to force Hh to turn on en, although enough Hh was produced to turn on dpp. Indeed, ptc LOF clones (which make cells think they are receiving Hh) are able to turn en on anywhere in the A region when a null ptc allele is used, but weaker LOF alleles only manage to turn en on near the A/P boundary [2834, 4136]. The idea that more Hh is needed to activate en than dpp is supported by the seriation of strata in the border

δ

zone (hh-on, en-on, dpp-on) and of radii at hhGOF A sites (rhh < ren < rdpp). Because concentration-dependent gene expression is the hallmark of a morphogen [865, 1655], it looks like Hh is a bona ﬁde morphogen after all and not just a short-range inducer [2448, 4848]. A gentle nudge from Hh would therefore coax a cell into Link 1, whereas a ﬁrmer push would force it into Links 2 and 3. The “Hedgehog Gradient Model” postulates that Hh activates different genes (e.g., en or dpp) at different thresholds (Ten > Tdpp) as a function of distance (near vs. far) from its source (usually the P compartment but potentially at ectopic A sites) [154, 4136]. Additional support for this model comes from varying the level of Hh in the P compartment [4136]: as the amount of Hh drops (when t.s. hhLOF ﬂies are raised at different temperatures), the band of en expression disappears before

CHAPTER SIX. THE WING DISC

139

FIGURE 6.1. Fate map of the wing disc and the regenerative potency of its fragments.

a. Fate map (abridged) of a mature right wing disc as per Bryant [524], except that dots are actual SOP sites [1925] and thick lines are prevein zones (1–5) [4189]. (See [834, 987, 1368, 3372, 4429] for further details.) The wing is darkly shaded, the heminotum (a.k.a. body wall) medium shaded, and the hinge and pleura lightly shaded. The wing disc is ∼300 µm across × ∼450 µm long [524] – roughly twice the dimensions of a leg disc (cf. Fig. 5.1). Like the 1st- and 2nd-leg discs (vs. the 3rd [3422]), the wing disc has a neural connection to the CNS [2128], but the nerve ﬁber enters along with the trachea (not shown) [3565]. Based on cell diameters in the wing pouch (mean of 10 counts along 25-µm transects in Fig. 8b of [350]; cf. scale bar), the disc would be 130 cells across × 190 cells long, but these are gross underestimates because the epithelium is highly folded. Vein 1 is the bristled part of the anterior wing margin [1741] (thin dashed line = posterior margin). The A/P compartment boundary is drawn as a thick dashed line. Directions (A, anterior; P, posterior; D, dorsal; V, ventral) are given in the compass at right. Bristle names (“a” and “p” = anterior or posterior members): DC (dorsocentrals), NP (notopleurals), PA (postalars), PS (presutural), SA (supraalars), SC (scutellars). The layout of SOPs preﬁgures the adult bristle pattern, except that scutellar SOPs rearrange (“p” moves posterior to “a”). In a and c–e, the asterisk marks the peak of regenerative potency as mapped surgically (cf. Fig. 4.5). b. Derivatives of the wing disc (shaded as per fate map). During evagination (illustration between a and b) the wing pouch expands, folds along the D/V line, and tucks its V side underneath (“N” = notum) [1311, 3374, 4189]. When the hinge region contracts [4509], its cells pack densely [3374] with some apoptosis [2849] to form a menagerie of elements [526, 1866, 4065]. This contraption allows the wing to twist so as to maximize the lift per stroke [1045, 1115, 4748]. Distal edge of the hinge is approximate [678]. The hinge region is genetically distinct from the blade per se because (1) it overgrows in response to excess Wg [459, 2252, 2254, 3088], and (2) it does not depend on Dpp signaling [157]. Indeed, wg expression in the hinge is induced by vestigial-on cells in the blade [2570]. Bristles (except along front edge) are omitted. c, d. Regenerative potency, assessed by γ-irradiation of young wing discs [3440]. c. Darker shading indicates higher frequencies (50–60, –70, –80, –90, –100%) of remaining markers in discs that exhibit deﬁciencies but no duplications. d. Darker shading denotes higher incidence (0–5, –10, –20, –60, –80%) of duplicated markers in discs that manifest duplications and deﬁciencies. Note that the same (central) region resists becoming deﬁcient (c) or duplicated (d). e. Path of the A/P compartment boundary in the peripodial membrane (PM, solid line) and columnar epithelium (CE, shaded line), as assessed by engrailed and patched expression [1133, 2072, 2216]. The regenerative peak (asterisk) falls between the A/P lines of the two layers (in top view). The D/V boundary in the peripodial membrane has not been charted so precisely [498]. The signaling role of this membrane in normal development is only starting to be explored [773, 1473, 3508]. f. Superposition of the entire A/P boundary (e) on the 1/4 (α, β, γ, δ) and 3/4 fragments whose regenerative behavior helped found the Polar Coordinate Model (cf. Fig. 4.6a). As indicated in the key (cf. Fig. 4.5), an unﬁlled arrowhead means that the fragment to its rear duplicates, whereas a solid arrowhead means that the fragment behind it regenerates. Given recent insights into leg disc regeneration (cf. Fig. 5.10c), it may be possible to ﬁgure out why quadrants duplicate and 3/4 pieces regenerate, but no unifying hypothesis is obvious based on the geometry alone.

the band of dpp expression, implying that en requires more Hh than does dpp. Similar studies indicate that Hh turns on two other genes – patched (which encodes its receptor) and knot (which evokes vein 3) – at thresholds between Ten and Tdpp (Fig. 6.3) [2894, 4136, 4478, 4479, 4539]. This “rainbow” of different qualitative states could emerge (as per Wolpert’s French Flag [4723]) from “contour lines in a gradient landscape” [3074], and the thresholds might stem from variable afﬁnities of target promoters for activator vs. repressor forms of Ci [154]. Disparate afﬁnities might explain why the Dpp band persists for as long as 24 h after hh function is shut off (by pulsing heatsensitive hhLOF mutants), while the Ptc band vanishes within only 45 min [642]. These facts do not prove a morphogen mechanism, however, because seriations of states can arise in other ways [1805]. In the “Signal Relay Scenario” [713, 714, 2816, 2817, 4591], for example, Hh would induce a second signal (En?), which then induces a third (Dpp?), and so on [1807, 2076, 2466, 3923, 4159]. The key difference between morphogen and

relay models is that the former demand direct action on target genes while the latter eschew it [3074, 3087, 3091, 4137]. A signal-relay mode of action [1607] (a.k.a. “signaling cascade” [91, 1144, 2192], “sequential induction” [2409, 2716, 3644], or “bucket brigade” [355, 2448]) was ruled out for Hh in the boundary zone by replacing the normal Hh with a membrane-tethered Hh construct [572, 4136]. Under these conditions, en and dpp both turn on only in A-type cells that touch hh-on cells – i.e., a single row of cells. (Coexpression of en and dpp is somehow tolerated despite the “En dpp” link.) If Hh were using En (or some other proxy) to create the dpp-on state, then two (or more) separate rows of cells should have appeared at the edge of the hh-on domain – the inner row expressing en and the outer row expressing dpp. Thus, Hh must be directly activating en and dpp. Ironically, a signal-relay scenario does apply from this point on. After Hh switches on dpp in neighboring cells, Dpp diffuses more extensively (as a second messenger) to organize pattern on a discwide scale.

140

IMAGINAL DISCS

brk 3

en 3-A

ci 3

dpp 3-P

dad 3

omb 3-A

spalt 3-P

wg late 3-P

al 3-P

tkv 3

knirps 3

vg 3-P

Dll 3-A

emc 3

gro

wg 2

w

gro

wg early 3

w

hh 3

vn 2 ?

ap 2-A

Dfz3 3

ac 3-P

rho 3-P

cut 3

vvl 3

hth 3-A

bs 3-A

A V

D P

Dpp turns ON omb and spalt at different thresholds While Hh speciﬁes fates over a small range (∼10 cells from its source), Dpp is evidently a morphogen for the wing’s entire A-P axis (∼50 cells in either direction within

the wing pouch) [2115, 2455, 3074, 4251]. Suppressing dpp causes small or missing wings [529, 3438, 4033]. The argument in favor of this “Dpp Gradient Model” rests largely on two genes that are activated by different levels of Dpp. Both genes appear to encode transcription

CHAPTER SIX. THE WING DISC

141

FIGURE 6.2. Patterns of gene expression in the wing disc. The ﬂow of control is from the selector genes en (upper right) and

ap (lower left), which deﬁne compartments, through dpp and wg (left), which specify orthogonal coordinates, to rho and bs (lower right), which dictate vein vs. intervein identity. Black areas chart mRNA (transcribed from endogenous or reporter gene) during 2nd instar (2), late-3rd instar (3), early pupal period (P) or adult stage (A). Shades of gray denote degrees of expression. Boxed pairs have roughly complementary patterns. Genetic interactions are indicated by connecting wires ( activation; inhibition; see text or sources cited at the relevant genes below). See also App. 7. Genes (“DO” = details omitted): ac (achaete; DO: other spots, asynchronies, and shape irregularities) [3689] , al (aristaless) [618] , ap (apterous) [361] , brk (brinker) [3978] , bs (blistered) [3145] , ci (cubitus interruptus) [1135], cut (DO: cut-on stripe is slightly thinner than wg-on stripe [2839] ) [988] , Dad (Daughters against dpp) [2867] , Dfz3 (Drosophila frizzled3) [3977], Dll (Distal-less; DO: pinching at edges of the wing pouch [618] ) [3242] , dpp (decapentaplegic; DO: gap in notal area [3764] and tapering to a point at future wing tip [4575] ) [4188] , emc (extramacrochaetae) [913], en (engrailed) [2954] , hh (hedgehog) [4228] , hth (homothorax) [157, 678] , knirps [2617], omb (optomotor-blind; DO: indentation at wing margin, depression at A/P border [4251], fade-out at edges, and proximal tapering) [3978] , rho (rhomboid) [4189] , spalt (DO: indentation at wing margin, depression in the 3–4 intervein [984, 4251], fade-out at edges, A/P asymmetry, and pupal details [4188]) [2867] , tkv (thick veins; DO: depression at A/P border [1327] and late-3rd instar expression in P compartment) [1674, 2457] , vg (vestigial) [678] (see [353] for pupal stages), vn (vein) [3928] , vvl (ventral veinless; a.k.a. drifter; DO: faint spot in notum) [703, 990] , wg (wingless) [2954] (cf. details about early stages [207] , pupal stage [834], hinge rings [678] , notum [1380, 3373] , and margin [3689]). Note the nesting of spalt’s major domain inside omb’s domain, which in turn is a subset of Dad’s zone [619, 2867]. This sort of successive subtraction may involve the layering of regional repressors.

factors that could convey Dpp’s signal to other echelons of genes downstream [3074]. 1. The optomotor-blind (omb) gene was found in a screen for ﬂies unable to use visual cues to navigate a maze [3367], and its LOF trait has been traced to neural defects in the optic lobes [3366, 3408]. Omb (974 a.a.) shares a T-box DNA-binding motif [3254, 3255, 4000] with vertebrate Brachyury [3365, 3366, 4001] and has been shown to bind DNA [3365]. While Dpp and Wg control omb expression in the wing [1626], omb is regulated by Hh in abdominal segments (sans Dpp or Wg mediation) [2302]. In both cases, the actions of omb are partly redundant with those of Scruffy [2302]. This redundancy may explain why omb LOF clones only cause defects when they cross the wing margin [1626], despite (1) omb’s endogenous expression in a wide swath of the wing pouch (Fig. 6.2) and (2) omb’s GOF ability to induce ectopic wings when expressed in the notum. Although omb assists Dpp in patterning, it seems not to help Dpp in promoting growth [1626] (cf. a reciprocal case [511]). 2. The spalt gene (“spalt” is German for “split”) was recovered based on its LOF embryonic lethality and head transformation [3152], which involves homeosis [672, 2101, 3737]. Along with its paralog spalt-related [221] located ∼65 kb away, spalt is activated by Dpp [986] starting in 2nd instar [4188] via a cis-regulatory region that is packed with discrete enhancers [220, 987, 2345] (cf. the en-inv paralog pair [1659]). Spalt (1355 a.a.) has 7 zinc ﬁngers (2 pairs and 1 triplet), 2 opa motifs, and C-terminal stretches of serine, alanine, and proline

[2346],

while Spalt-related (1263 a.a.) has 8 zinc ﬁngers and similar homopolymer stretches. A fragment of Spalt-related containing 3 conserved zinc ﬁngers binds the DNA sequence TTATGAAAT [221], although its endogenous target genes remain unknown [987]. In 1996, two teams – Thomas Lecuit et al. in ¨ Heidelberg [2455] and Denise Nellen et al. in Zurich [3074] – showed that omb and spalt are targets of Dpp in the wing disc. The “Dpp omb” and “Dpp spalt” links were demonstrated by LOF-GOF manipulations of the Dpp pathway: (1) omb and spalt expression vanish in dpp LOF discs, and similar effects are seen with tkv LOF or Mad LOF clones; (2) omb-on and spalt-on domains expand to ﬁll the A-P axis when dpp or tkv Q253D (encoding a constitutively active Tkv receptor) is expressed widely (viz., ubiquitously or along the future margin). Dpp’s effect on omb and spalt must be direct (vs. via signal relay) because although both genes can be activated outside their normal areas by dpp or tkv Q253D , only dppGOF clones force their expression into surrounding wild-type tissue [2455, 3074]: tkv Q253D clones turn omb and spalt on exclusively within the conﬁnes of the clones. If diffusible second messengers existed, then they should have evoked omb-spalt expression outside the clones (nonautonomy vs. autonomy) [3999]. Dpp appears to turn omb and spalt on at different concentrations because the endogenous stripes are of different widths: the omb-on stripe is broader than the spalt-on stripe (Fig. 6.2). The inferred ranking of their thresholds (Tspalt > Tomb ) is conﬁrmed by the concentric circles of target gene expression that attend ectopic

142

IMAGINAL DISCS

vg 1 2 3 4

Dll

A P

5

? Emc

ac

A V

D P

43 cells

100 µm

Wg

dpp ptc en knot

vg

1

omb spalt

2 Hh

3 4

A P

Dpp

5

D V FIGURE 6.3. Morphogen gradients that dictate wing cell fates (cf. Table 6.1). The wing pouch from a mature right wing disc

(above left) is idealized as an oval (cf. Fig. 6.1), with solid lines (A/P or D/V) between compartments. Dashed lines denote veins 2–5 (vein 1 = part of the margin). Scale bar applies to the enlarged oval. Black triangles around the oval are gradients of Hh, Wg, or Dpp, although Hh is also present in the P region (dotted rectangle). Outer bars indicate extents of target gene transcription, although en is also on in P cells (dotted bar) and ptc is on at a basal level in remaining A cells (not shown). For gene abbreviations, see Fig. 6.2 or below. For additional details and perspective, see App. 7. Hh diffuses into the A region [4228] where it turns on (1) dpp [4136, 4479], (2) patched (ptc) [2728, 4136, 4479, 4539], (3) knot (a.k.a. collier) [1840, 2894, 3077, 4479], and (4) en [4136] (late-3rd instar only [350, 4228]) at successively higher thresholds (although thresholds for ptc and knot are comparable). Because en dpp [1647, 2980, 3747, 4229], dpp only remains on in the front 2/3 of the Hh gradient during late-3rd instar [4136] (but see caveats in App. 7). Light shading denotes suppression. Ptc’s level should also drop where it overlaps en-on [277, 350, 2728, 3372] because en ci and Ci is needed for ptc transcription [2832, 3747, 4229] (cf. Figs. 5.6 and 5.7). Why it does not is unclear [4539]. At the same threshold as dpp, Hh also turns on master of thickveins (not shown) [1327]. At a lower threshold than any of those listed above, Hh turns on the Iro-C genes ara and caup (not shown; cf. Fig. 6.11) [320, 1537, 2993]. Cells in the dpp-on stripe make Dpp, which diffuses to turn on (1) vg [2217, 2219], (2) omb [2455, 3074], and (3) spalt [2455, 3074] at successively higher thresholds. Cells at the D/V boundary make Wg, which diffuses to turn on (1) vg [678, 2217, 3091, 4849], (2) Dll [166, 1040, 3089, 3091, 4849], and (3) achaete (ac; also scute, not shown) [166, 912, 3689, 3982] at successively higher thresholds, though Dll and vg respond in a graded manner. So do Dfz3 (activated) [3977] , vvl (repressed) [703, 990, 995], and the Iro-C genes ara and caup (repressed) [1537] (not shown). The ac stripe splits into two bands (each ∼4 cells wide; cf. Fig. 6.8) [2079, 3689, 3690] apparently due to Cut ac [988], possibly via Emc [913] or Mβ [871] or both [205]. Cut was once thought to be a Wg target [352, 353], but the link is indirect [2839, 3089] via Vg [3936] and Scalloped [2940]. Oddly, Wg also turns on omb (not shown) [1626] via the same cis-enhancer that is under Brinker-mediated Dpp control [3978]. The overall diagram is adapted from [3087, 4137], but amended for vg regulation [703, 2217], with speciﬁc pathway data from Hh [2992, 2993, 4136, 4478, 4479, 4539], Dpp [2455, 3074, 3406, 3972], and Wg [4849]. Cell dimensions (scale bar) reﬂect the mean of 10 counts along 25-µm transects in Fig. 8b of [350].

CHAPTER SIX. THE WING DISC

dppGOF clones: omb and spalt are activated at different ranges (rdpp < rspalt < romb ) [3074]. Lecuit et al. veriﬁed this ﬁnding after initially obtaining contradictory results [2455, 3999] that were attributable to not allowing enough time for the “halos” to materialize [2457]. When the dpp-on stripe and the en-on P compartment are also taken into consideration, the endogenous on or off states of omb and spalt would subdivide the A-P axis into 7 zones [3074] – each with a unique set of transcription factors (Ci-155 rules the dpp-on zone). Using a binary code for the sets of on/off states (En, Ci-155, Spalt, and Omb from left to right), the zones (A to P) are 0000, 0001, 0011, 0111, 1011, 1001, and 1000 (cf. Figs. 5.7 and 6.3). Dpp concentrations evidently specify different structures along the A-P axis because the weaker spalt expression in tkv LOF (or saxnull ) clones is associated with shifts in fate (in terms of vein location or marginal bristles) [3972]. The autonomy of the shifts implies that the cells are less able to “hear” the Dpp signal, so they “think” they are farther from the Dpp source than they actually are (cf. a similar case for Hh [2993]). Nellen et al. make a clever case against any other A-P gradients of fate-determining factors (e.g., Gbb [1674, 2203, 4610]) in the wing disc. If such factors existed, they argue, then the omb-on and spalt-on “halos” around ectopic dppGOF clones should be skewed in the direction of those other gradients. In fact, however, the halos are uniform in width all around the clones, regardless of where the clones are located in the disc.

Dpp regulates omb and spalt similarly despite clues to the contrary Lecuit et al. examined omb and spalt expression in dppGal4:UAS-dpp wing discs, where dpp is upregulated in its usual stripe. These discs are grossly (∼4x) widened along the A-P axis (except in the notal area) because Dpp is a potent mitogen (cf. Ch. 5). On the simple assumption that spalt and omb are direct read-outs of high or low Dpp levels, respectively, both bands (spalt-on and omb-on) should widen to the same extent (viz. ∼4x), but neither band does so. The spalt-on band widens by ∼1.3x, whereas the omb-on band retains normal size. The net result is that the two bands become congruent. On this basis, Lecuit et al. devised an “Omb Memory Hypothesis.” Expression of spalt, they argued, is a true read-out for Dpp (ignoring the 1.3x vs. 4x discrepancy) because spalt is continually sensitive to Dpp (cf. the Cabaret Metaphor, Ch. 4). In contrast, expression of omb would be sensitive to Dpp only early in development.

143

The omb-on state would “ratchet up” early [1657, 3999] and stay on heritably in all descendant cells, regardless of their later locations in the Dpp gradient [4575]. (Assuming that Gal4-driven Dpp arises after omb-on is recorded, spalt’s domain could theoretically expand without affecting omb.) This hypothesis cannot be true, however, because omb needs Dpp continuously [571, 4849]: omb expression vanishes in tkv null clones until extremely late in 3rd instar [3074] (cf. similar omb downregulation in late Mad LOF clones [2455]). In 1998, a simpler explanation was proposed by Haerry et al. [1674] and Lecuit and Cohen [2457] based on the fact that Tkv is expressed intensely just outside the omb domain but weakly inside it until late 3rd instar (Fig. 6.2) [512, 980]. This complementarity could explain omb-spalt congruence if high levels of Tkv were acting as a barrier for Dpp diffusion. In that case, the excess Dpp in dpp-Gal4:UAS-dpp discs would push the edges of the spalt-on domain out to the Tkv barrier but no farther, whenceforth the spalt-on and omb-on domains would coincide. Several lines of evidence support this “Tkv Barrier Hypothesis”: 1. In dpp-Gal4:UAS-dpp discs, the tkv-on domain continues to “hug” the omb-on domain just as it does in wild-type discs (and just as it should if it is a barrier), despite excess tissue growth outside the omb-on area [1674]. 2. Overexpression of a wild-type tkv transgene in the wing pouch causes A-P shrinkage of both the omb-on and spalt-on bands [1674], as if the excess Tkv is impeding the outward movement of Dpp. Consistent with this inference, the omb-on band becomes nonuniform, with intense expression along the dpp-on stripe and lower expression peripherally. 3. Dramatic evidence for an effect of Tkv on Dpp diffusion is seen in discs where en-Gal4 drives a wild-type UAS-tkv transgene [1674]. Because the en-on domain in wild-type discs abuts the rear edge of the dpp-on stripe, en-Gal4:UAS-tkv discs should acquire a new Tkv barrier just behind the dpp-on stripe. Indeed, expression of omb in these discs is intense precisely there, as if posteriorly directed Dpp is hitting a wall and increasing in concentration.

Dpp does not regulate tkv in 3rd instar despite clues to the contrary The complementarity between omb-on and tkv-on domains in wild-type discs (Fig. 6.2) could easily arise

144

IMAGINAL DISCS

4 En

Ci

?

En 0

0

A-type Ptc (listens)

1

Dpp

and

5

3 1

2

dpp en dpp hh

A B P 5

d 0

0

En 1 1

1

and

5 Pattern

Dpp

from a “Dpp omb tkv” cascade. The “omb tkv” suppression cannot be total, however, because a minimum amount of Tkv is needed for omb expression in its endogenous domain [3074], and Tkv is also required there for cell proliferation [569, 3972]. Lecuit and Cohen argue that some sort of “Dpp tkv” link (with or without omb as an intermediary) must exist because tkv expression is suppressed near clones that ectopically express dpp [2457]. However, the Gal4 driver they used

1

P-type 1

sion

1

4

1

0

diffu

1 0

2 3 4

1

A-type

1

1

e

(mute)

0

3

f

(deaf)

Hh Hh En en Dpp

En + Inv Hh Dpp

b

0

0

1

and

1 2 3 4 5

A-type

1

2 Dpp

0

Hh (speaks)

P-type

Inv

(mute)

c

a

(“C765”) cranks dpp expression far above physiological levels, and the discs were not examined until 48 h after clone induction. Thus, it is possible that the reduced Tkv is an artifact of the “industrial strength” Dpp or a distant side effect of prolonged exposure to Dpp (e.g., induced expression of omb or other genes that could confer new cell identities). Indeed, other experiments argue against any regulation of tkv by either dpp or omb.

CHAPTER SIX. THE WING DISC

145

FIGURE 6.4. Circuitry that enforces A vs. P identities of wing cells and causes expression of dpp at the A/P interface. Abbreviations: Ci (Cubitus interruptus), Dpp (Decapentaplegic), En (Engrailed), Hh (Hedgehog), Inv (Invected), Ptc (Patched). a. Generic wing disc cell (cf. Fig. 2.7 for icons) showing the main circuit that operates throughout disc development (light gray), plus a “plug-in” module that works only in late-3rd instar (dark gray) [1647]. Numbers refer to links in b. b. Major links, redrawn using epistasis symbols (cf. Figs. 5.6 and 5.7). Link 2 (receipt of Hh signal activates en) only becomes operative in late-3rd instar, when the en-on state spreads into the A compartment to ﬁll the zone marked “B” in the wing diagram (cf. Fig. 6.7d) [350]. Because Link 2 requires more Hh than Link 1 [4136], the band of dpp-on cells induced by Hh extends farther anteriorly than the band of en-on cells, and Link 3 then turns off dpp where these bands overlap (cf. Fig. 6.3). Link 5 is a “safety switch” that prevents a runaway cycle of en (Link 2) and hh (Link 4) activation that would convert all cells to a P-type state (cf. Minotaur Scenario; Fig. 5.10c-e; Ch. 5). Link 5 can be overridden by excess Hh (not shown) [2992], so an analog pressure clamp would be a more apt analogy than a digital switch. Literally, the clamp is probably the receipt of a maximal Dpp signal by Tkv-Punt receptors, but it might instead be an inability of dpp-on cells to turn on en. All we really know for sure is that en-on states cannot traverse dpp-on tissue. Link 4 is disabled in the B zone [2992] because hh is more sensitive to repression by Ci-75 than to activation by Ci-155 [2980]. In any event, the plug-in module is incidental to the grand scheme and is omitted from further consideration. c–e. Schematics of cells in 3 different districts of gene expression along the A-P axis before late-3rd instar. “A” denotes the anterior A compartment beyond the range of diffusible Hh, “B” is the border zone (of A-type cells) that responds to Hh by forming a stripe of dpp-on cells, and “P” is the P compartment. States of variables (cf. a) are recorded as “1” (present) or “0” (absent), although thresholds do matter (discussed above). Black circles indicate decisive factors. In plain English, the logic of the main circuit is as follows. If en is off (c and d), then the cell takes the Ci route, deploys Ptc (Hh’s receptor), and adopts A-type identity, although an agent other than Ci may mediate the latter. If en is on (e), then the cell secretes Hh and turns on inv, which causes P-type identity. One-way signaling between Hh-secreting “speakers” (e) and Ptc-competent “listeners” (d) establishes a dpp-on stripe in the B zone, which then secretes Dpp (cf. Fig. 6.5). Although P cells are bathed in Hh, Link 1 cannot work there because (1) they lack Ptc and so are deaf to Hh [328, 4229], (2) they lack Ci (due to “En ci”) and so cannot activate dpp (“Ci dpp” link not shown) [1827, 2832, 4229], and (3) En represses dpp directly by binding its cis-enhancers [2980, 3747]. Because cell states are set by whether en is on and whether Hh is received, these variables (black circles in c–e) act like a binary code, where the ordered pairs (en on?; Hh bound?) are 00, 01, and 10. These codes deﬁne the A, B, and P identities. f. Proﬁles of protein distributions (black bars or triangles) before late-3rd instar relative to districts of gene expression, which are demarcated on a realistic wing at left and a schematic at right. Rear dashed line is A/P boundary (after [497]). Invected (under En control) lets A vs. P cells interpret symmetric Dpp gradients differently to make an asymmetric pattern (i.e., veins 1–3 vs. 4–5) [3934, 3935]. Circuitry in a is based on [643, 1647, 1659, 3935, 4229, 4848], and the gradients in panel f are adapted from [4229, 4734]. The logic of this circuit is dramatically conﬁrmed by using dpp-Gal4 to drive UAS-hh. These conditions entrap the system in an “inﬁnite loop”: every time an A-type cell is instructed by Hh to turn on dpp (d), it must now also turn on hh, leading to another round of Hh signaling that enlarges the hh-on area still farther. The outcome of this robotic iteration is a grotesque multiplication of bristles and sensilla that are normally limited to the A/P boundary [2992]. N.B.: Input/output ports (raised rectangles) on the cell surface are drawn at arbitrary apical-basal levels (apex denoted by microvilli). The “en hh” link is written in gene symbols because – unlike “En dpp” – it may be indirect [224] since En is normally a repressor (cf. Fig. 5.7 legend). The role of inv is unclear because null alleles have no apparent effect [358]. For other examples of “safety switches” that override morphogen inputs, see [2176, 3733].

1. Ubiquitous overexpression of dpp (using a moderate Gal4 driver) does not downregulate tkv, nor does a constitutively active Tkv receptor (Tkv Q199D ) when it is expressed similarly [1674]. These results argue against a “Dpp tkv” link, and another aspect of these same discs refutes an “omb tkv” link. To wit, when the omb-on band expands, it overlaps the normal tkv-on domain in the distal half of the disc, but does not affect tkv. 2. During late 3rd instar, the wild-type tkv-on pattern changes so there is now extensive overlap between omb-on and tkv-on domains in the P compartment [2457]. This overlap again refutes an “omb tkv” link.

3. Clones of dppGOF cells show no lower Tkv-dependent phosphorylation of Mad within or around the clone, whereas hhGOF clones do (implying that “Hh tkv”) [4251]. 4. The wings of ﬂies whose Hh is replaced by a membrane-tethered Hh construct have remarkably few defects (∼50% linear dimensions, missing 3/4 intervein; Fig. 6.6h), considering that their dpp-on stripe is only 1 cell wide (vs. the normal ∼5-cell width; Table 6.1) [572, 4136]. Robustness of this sort implies a circuit wherein the Dpp output per cell varies as an inverse function of the number of Dppsecreting cells.

146

IMAGINAL DISCS

TABLE 6.1. GRADIENTS THAT ESTABLISH CELL FATES IN THE WING PRIMORDIUM*

Feature

Hedgehog Gradient

Decapentaplegic Gradient

Wingless Gradient

Ligand (mature secreted fragment)

Hh = glycosylated polypeptide (214 a.a. piece of 471 a.a. precursor that cleaves itself ) covalently linked to cholesterol [255, 2705, 3432, 3434]. PC [2467, 2832, 4227, 4228, 4254].

Dpp = glycosylated polypeptide (132 a.a. piece of 588 a.a. precursor), which forms S-S linked dimers [1427, 3224, 3244].

Wg = glycosylated polypeptide (451 a.a. piece of 468 a.a. precursor) [175, 3561, 3599].

AC stripe that abuts the A/P boundary [754, 2739]. On average, the stripe is ∼5 cells wide [4136, 4479], but it varies from ∼2–8 cells in width and even dwindles to zero at the wing margin [3497]. Unknown. Needs proteoglycans (including Dally) [2004, 2556, 3038] and clathrin (implying endocytosis) [1546].

Stripe that straddles the D/V compartment boundary [163, 177, 882, 4683]. Wg is expressed intensely in a band ∼4 cells wide [882, 3091, 3689, 4138].

Source of ligand (“AC” and “PC” are anterior and posterior compartments)

Mode of ligand movement (possibilities including free diffusion [2780], transcytosis [2843], and cytonemes [3507])

Unknown [786]. Needs proteoglycans (but not Dally) [277, 4275]. Hh’s cholesterol tail suggests that Hh may ride “lipid rafts” [1896, 3946], but it could be a “handle” for passing Hh from cell to cell via Ptc [572]. Ptc colocalizes with Hh along the apical-basal span of each cell as punctate dots that suggest vesicles [572]. Ptc is mainly in lateral membranes and undergoes endocytosis [644, 731, 1554].

Effect of receptor on ligand movement

Ptc impedes Hh movement [277, 754, 755, 2074].

Tkv impedes Dpp movement [1674].

Effect of ligand on receptor gene expression

Hh

Orientation of gradient

A-P axis [4136, 4479, 4539].

A “Dpp tkv” link can be forced artiﬁcially [2457] but is unlikely to operate in nature [1674]. A-P axis [3074].

Unknown [1910]. Needs proteoglycans (including Dally) [2556, 3038, 3561]. Different parts of the Wg molecule cause receptor activation vs. intercellular transport [273, 1055, 1779]. Uptake into vesicles by receiving cells occurs in the absence of Fz and Dfz2 (implying unknown nontransducing binding proteins) [2984]. Dynamin (and hence endocytosis) is needed for Wg transport in embryos [2900], but Wg can move extracellularly in discs without it [4138]. None [595]. Dfz2 retards Wg degradation and increases cells’ sensitivity to Wg signal but does not affect Wg movement. A “Wg Dfz2” link operates in wing discs [595] but is not likely to play any role in patterning [734]. D-V axis [4138, 4849].

Farthest effects (deduced from LOF-GOF studies and probes of target gene expression)

Direct effects (ignoring secondary consequences of Dpp) extend ∼10 cells [277, 786, 2728, 2993, 4136] from A/P boundary to ∼ vein 3

Entire A-P axis of the future wing (∼50 cells in either direction from A/P line) [3438, 4032, 4251, 4265].

Entire D-V axis of the future wing (∼40 cells in either direction from D/V line) [1910, 3977, 4849].

ptc [642, 1841, 1982, 2074].

[572, 1837, 2992, 4136, 4188]

(although Hh itself does not induce vein 3 [364]). A similar impact radius is seen around ectopic hh-GOF clones [572].

CHAPTER SIX. THE WING DISC

Range of ligand detected (by antibody staining) away from its source**

2–4 cells [277, 572, 4228].

Target genes (“high,” “medium,” or “low” refer to thresholds for activation)

en (high [4136], late-3rd instar only [4228]), ptc (lower than en [4136] but debatable as to whether it is high [572, 2728, 4479, 4539] or medium [277] or low [4136]), kn (medium? [2894, 4479]), dpp (low [4136, 4479]), Iro-C genes ara and caup (minimal [320, 1537, 2993]).

147

∼ 35 cells (80 µm) from A/P stripe of dpp-on cells, as assessed by a GFP-tagged Dpp construct [1169, 4265]. This range nearly spans the pouch. Dpp traverses ∼4 cells per hour [1169]. spalt (high [2455, 3074]), optomotor-blind (medium [2455, 3074]), vestigial (low [2219]).

∼ 25 cells from D/V boundary (≈ the entire breadth of the pouch) is the maximum reported [595, 3691].

achaete and scute (high [882, 912, 3374, 3689, 3982]), Delta (high [595, 988]), Distal-less (medium [3091, 4849]), vestigial (low [3091, 4849]).

*See Figure 6.3 for disc geometry. Ranges refer to mature discs. Gene abbreviations: dpp (decapentaplegic), en (engrailed), hh (hedgehog), kn (knot), ptc (patched), wg (wingless). N.B.: The ability of molecules to exert effects at concentrations that are undetectable by antibodies (e.g., Hh [4136]) is not unique to these morphogens [4489, 4849]. Historically, such negative results confounded the analysis of Notch (cf. Ch. 2), Ci [155, 4539, 4540], and Mad [3095, 3498], as well as the axis and gap gene products [1946, 3248]. A current example is neuralized, which is not detectably expressed in PNC cells but must directly affect them because its null allele autonomously transforms them into SOPs [2387] (but see [2387] sequels for evidence to the contrary). **Until 2000, the diffusion range of Dpp was not directly known [3406] due to lack of suitable antibodies [1546, 1807, 3087] The use of Green Fluorescent Protein (GFP) to tag Dpp has been a breakthrough [1169, 4265], and the Dpp-GFP construct is biologically active. Nevertheless, its molecular weight exceeds that of the native Dpp ligand, so its concentration proﬁle and turnover rate may be misleading. Diffusion ranges for other ligands are crudely estimated to be ∼10 cells for Argos [1294, 2331, 4035], ∼3 cells for Scabrous [186, 2461], and ∼4 cells for Spitz [1288, 1337].

Indeed, cells in the dpp-on stripe must be using Tkv to monitor the intensity of Dpp signal because (1) the dpp-on stripe disappears when a tkv GOF transgene is expressed ubiquitously (see also the small wings in dpp-Gal4:UAS-tkv ﬂies [4251]), and (2) the dpp-on stripe doubles in width when a tkv LOF state is created along the stripe (via a ptc-Gal4 driver and a dominantnegative UAS-tkv DN ) [1674]. This sort of modulation could not work with a “Dpp tkv” link because the latter would prevent cells from sensing the absolute amount of Dpp. Indeed, all cells in the wing pouch (not just the ones in the dpp-on stripe) must be sensing an absolute level of Dpp because clones with excess Tkv ratchet their identities (e.g., from omb-on to spalt-on), as if they are “hearing” a stronger Dpp signal [2457]. Tkv-mediated autoregulation may also explain the ability of the dpp-on state to sustain itself for ≥12 h when deprived of Hh [2992]. Despite the above caveats, the idea that Dpp affects its receptor’s expression has become a gospel truth [1327, 1546, 3347, 3406, 4137, 4251], and derivative models are starting to be based on it [977, 3225]. The “Dpp tkv” link is seductive because it would explain why mitoses can be uniform throughout each disc [1134, 1142], even though the major

mitogen (Dpp) is concentrated along one (A/P) line [1839]. To wit, if ligand and receptor gradients are opposed to one another, then all cells could “hear” the same level of Dpp regardless of their distance from the source – like a rock concert where fans standing closest to the band tend to be the deafest. Two additional facts refute this “Rock Concert Scenario.” If a “Dpp tkv” link were operating, then increasing the amount of Dpp via dpp-Gal4:UAS-dpp should have no effect because all cells should become deafer (i.e., reduce their Tkv) as the dpp-on stripe raises its volume (i.e., increases Dpp output). In reality, however, the disc quadruples in width. One way out of this corner would be to assert that the “Dpp tkv” link only works up to a certain level, above which cells hear more Dpp and multiply faster. In that case, proliferation should still be uniform throughout the disc, but here the second contradiction is encountered. The area where omb and spalt are expressed is virtually unaffected, while the outlying areas balloon to huge proportions [2455]. Such behavior implies a step function rather than a gradient, and Tkv does have a step function proﬁle [4265] (Fig. 6.2; cf. the Tkv Barrier Hypothesis). Thus, although Tkv mediates both the mitogen and morphogen

148

effects of Dpp, it does not suffer the kind of modulation that Dpp exerts on Dad or brk (cf. App. 6). Putting tkv under the control of “prepattern genes” that are independent of dpp makes sense because it would allow evolution to orchestrate the shapes of adult organs by targeting more or less Tkv to speciﬁc parts of a disc at speciﬁc times, thereby tinkering with growth rates in a balkanized way. The “programming language” of morphogenesis undoubtedly involves such tricks [1165, 4114]. In 2001, one of the prepattern genes that controls tkv was conﬁrmed. It is hedgehog. When Hedgehog diffuses into the A compartment, it turns on a gene called master of thickveins (mtv) [1327], which suppresses tkv. The “Hh mtv tkv” chain operates in parallel with the “Hh dpp” link. How tkv is regulated in the rest of the pouch is unclear because mtvnull clones have relatively little effect on tkv levels beyond the A/P border zone [1327]. Many other riddles persist about how Dpp functions as a mitogen. For example, if each section of the dpp-on stripe is needed for normal growth [3438, 4032], then why do outgrowths arise only when ectopic dpp-on stripes cross the D/V line [1839, 4848] (see below)?

A vs. P identities might explain how a straight A/P line emerges The dpp-on stripe establishes two gradients along the A-P axis. To a ﬁrst approximation, the A gradient spans the A compartment and the P gradient spans the P compartment. In reality, the A/P gradient interface (“AG/PG”) is offset from the A/P compartment boundary (“AC/PC”) by ∼6 cells because the peak expression of dpp is in the middle of the 5-cell wide dpp-on stripe, and that stripe is separated from the AC/PC line by the 3–4-cell wide band of en-on A cells (Fig. 6.3). The implications of this imperfect registration are addressed later. How can back-to-back gradients that use the same morphogen produce different patterns of veins, bristles, etc., in the adult wing [904, 4734]? The obvious answer (cf. Ch. 4) is that engrailed acts a “selector gene” [1358]. To wit, en-on P cells would adopt P-type competence that makes them interpret Dpp levels in one way, while en-off A cells adopt (by default) A-type competence that makes them interpret Dpp levels in a different way (cf. Fig. 4.3b) [643, 1628, 1838, 2441, 2930]. Enforcement of competence states would be a second duty of compartments [231, 1807, 3862], aside from their creation of a morphogen stripe via the Deaf-Speakers/Mute-Listeners Trick (cf. Ch. 5) [328, 4848]. By using this genetic gadgetry, an

IMAGINAL DISCS

asymmetric pattern can thus be built on a symmetric scaffold. Indeed, the effects of en1 were a cornerstone for Garc´ıa- Bellido’s Selector Gene Hypothesis [1355, 1358]. This allele partly transforms P compartments to look like A ones and thus causes asymmetric patterns to become symmetric (e.g., the foreleg acquires a second sex comb in mirror image to the normal one [439]). The en gene was long suspected of being involved in transducing rather than sending positional information because it essentially behaves autonomously in mosaics. For instance, small patches of en1 tissue can form a sex comb on the P (wrong) side of the leg [4343] and triple-row bristles on the P (wrong) edge of the wing [1378]. Once en was cloned in 1985 [1031, 1247, 2356, 3429] and shown to encode a DNA-binding transcription factor [2047, 3719], the case for its involvement as a “switch gene” was bolstered. The case was further strengthened when en was found to be expressed coextensively with P compartments (as deﬁned by cell-lineage restrictions) of thoracic discs [495, 1697, 1963, 2307]. This congruence was expected because en offered a way to neatly explain the straightness of the wing’s A/P boundary. The A/P line in the adult wing runs for hundreds of cells without wavering more than one cell diameter [350, 1078, 3441]. It is also straight across entire wing and leg discs through early 3rd instar [569, 2728, 3764], although it later manifests kinks [495, 497, 1022, 1833], possibly due to being “pushed” by uneven growth [497]. Speculation about en as a boundary-straightening agent began in 1975. Gines Morata and Peter Lawrence proposed that A and P cells have different afﬁnities due to expression (P) or nonexpression (A) of en [2441, 2928]. They imagined that a line forms between A and P cells in the same way that oil and water segregate to form an interface (viz., via homophilic sorting). The evidence for this “Selector Afﬁnity Model” is reminiscent of leg clones with improper P-D states (e.g., Dll [1561] or hth [1573]; cf. Ch. 5). 1. “Xenotopic” (i.e., located in alien territory) en clones can cross the A/P line: (1) crossing (P-to-A) by Atype en1 clones [2441, 2927, 2928] or enLOF clones [2306, 2447] and (2) crossing (A-to-P) by P-type enGOF clones [4848] (cf. Polycomb LOF [4327]). This strategy could correct sporadic mistakes so as to maintain separate pools of signaling vs. receiving cells [2677], although it is unclear how lineage restraints are enforced in late-3rd instar when en-on cells exist on both sides of the A/P line [354].

CHAPTER SIX. THE WING DISC

2. Xenotopic en clones far from the A/P line minimize contact with surrounding tissue by adopting a circular shape and smooth outline: (1) A-type en null inv null (but not en1 [1639, 2441]) clones in the P region [4229], (2) P-type enGOF clones (which have 2x normal cell density [4848]) in the A region [1078, 4848], and (3) polyhomeotic LOF (en-on) clones in the A region [3512]. 3. Xenotopic en clones far from the A/P line can form vesicles in an apparent attempt to leave the epithelium (cf. sorting of wing and haltere cells [2924, 3875]): (1) A-type enLOF clones in the P region [2447, 2927] and (2) polyhomeotic LOF (en-on) clones in the A region [3512]. However, A-type en1 bristles in secondary (P-side) sex combs do not leave the P territory in mosaics [4343]. Homophilic sorting implies A- vs. P-speciﬁc adhesion molecules [493], but none has so far been found [942, 1839], although En does control expression of Connectin and Neuroglian in the CNS [3925]. Generally speaking, cell identities must be implemented by echelons of “realizator” genes that are downstream of selector genes [1225, 1226, 1358, 1367, 1924], but only ∼5 possible realizators for en have surfaced thus far [3457].

But the A/P line appears to straighten via a signaling mechanism Notwithstanding all the supportive evidence, en’s status as a selector gene was controversial for many years [354, 1635, 1636, 1837] due to (1) neomorphic idiosyncrasies of en1 [852, 1138, 1636, 2306], (2) the inability of bona ﬁde LOF alleles to mimic its mirror-image syndrome [852, 1636, 2447], and (3) expression of en in A-type cells at the A/P border [4539]. A hidden confounding factor all along, it turned out, was an adjacent paralog – invected (inv) [842] – whose function partly overlaps that of en [1659, 3925]. Only when both of these homeobox genes are inactivated is it possible to achieve complete P-to-A transformations (Fig. 6.6) [4229]. To the extent that en and inv act redundantly, they should have similar effects when misexpressed in the A region. In fact, their GOF phenotypes differ considerably: a viable invGOF mutant has nearly perfect P/P symmetric wings (A-to-P homeosis) [3934, 3935], while enGOF A clones cause milder A-to-P conversions [4848]. Moreover, invGOF has virtually no effect on the expression of hh, dpp, ci, or ptc [3935], whereas enGOF alters them dramatically [1647, 4848]. Evidently, a division of labor exists: inv’s duty is to implement P identity downstream of en (“en inv P identity” [1647, 4229] but see [358]), whereas en has two duties: (1) to activate inv, and (2) to

149

activate hh so as to trip the Hh-Dpp relay that patterns the wing (“en hh dpp-on stripe at A/P boundary”) [1837, 4229]. Other genes aside from en must keep inv on in the P region because ennull clones do not mimic the Pto-A homeosis of en null inv null clones [4229]. In 1997, the orthodoxy of the Selector Afﬁnity Model was shaken by a heretical discovery. Clones of smo null cells can cross from A to P without ever changing their en state [364, 2435, 3627], and a similar A-to-P crossing was later seen with fu LOF clones [3177]. Some involvement of the Hh pathway with cell afﬁnities had earlier been suggested by the round shape of A clones (far from the A/P boundary) that secrete Hh (ci null [1078]) or “think” they are receiving Hh signal (DC0 LOF [2058, 2072, 2491, 2533, 3238], ptc LOF LOF [642, 754, 1078, 2834, 3372, 3627], or slimb [2856]), and the A-to-P null shift of smo clones can be halted by activating the Hh pathway downstream of Smo (via DC0 LOF ) [3627]. These results imply that the conﬁnement of A-born cells to A compartments and P-born cells to P compartments has nothing to do with A-type (en-off ) vs. P-type (en-on) states per se. Rather, the segregation appears to be enforced by a cohort of dpp-on A cells near the A/P line. Any A cell can join this “ fraternity” if it turns dpp on, which will happen naturally as A cells jostle close to the A/P line and encounter enough Hh (cf. the Cabaret Metaphor, Ch. 4). Likewise, A cells can depart by turning dpp off, which happens as growth pushes dpp-on cells too far from the P compartment, so the dpp-on stripe itself is not a cell-lineage compartment. As the examples below illustrate, this “Border Guard Model” [364, 1839] can explain the old phenomena as well as the Selector Afﬁnity Model, plus it accommodates the new results that the latter cannot (Fig. 6.5): 1. Crossing the A/P border from P to A. Given that a dppon state is a passport into the dpp-on stripe, while dpp-off means banishment thence, P cells never cross the border because their en-on identity prevents them from turning dpp on (en dpp). In an LOF en clone, the P-born cells acquire passports because the turning off of their en gene lets them hear the Hh signals all around them, whereupon they turn on dpp. If they are close enough to the A/P line, then they can enter and move farther into the A region by the exit route that A cells normally use. 2. Crossing the A/P border from A to P. Banishment from is imposed on any member whose dpp gene gets turned off. Such a switch will happen in border clones whose en gene is artiﬁcially turned on (enGOF dpp), but it can also happen when the Hh transduction pathway is shut off (e.g., smo null )

150

IMAGINAL DISCS

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

en - OFF en - ON

A en

P GOF

P

A

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P Selector Affinity Model Border Guard Model A

A

A

A

A

A

A

A

A

A

A

A

B

B

B

B

A

A

A

A

A

A

A

A

B

B

B

B

B

A

A

A

A

A

A

A

A A null

A

A

B

B

B

B

B

B

B

B

B

B

B

B

B

B

A dpp - ON

B

smo

A

A

A

A

A

B

A P

A P

A P

A P

A P

A P

A P

A P

A P

A P

A P

A P

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

P A

Hh

A P

FIGURE 6.5. Alternative explanations for why cells cannot normally leave the compartment where they are born. Part of the A/P

interface is shown in each panel, and cell types are enlarged at right. According to the Selector Afﬁnity Model (above) [904, 1369, 2441, 2928], the on or off state of a cell’s engrailed (en) gene “selects” whether it adopts P- or A-type identity. A-type cells use one kind of homophilic adhesion protein (round hook), P-type cells use another (zigzag hook), and A-P binding is disfavored. Only when cells switch identities – e.g., the shaded enGOF A clone – can they cross the A/P line (gray bar at right). In this schematic, one of the clone’s cells has already crossed. The Border Guard Model [364, 1839, 3627] postulates a unique adhesion protein (square hook) for A (en-off) cells within a certain range (∼5–10 cells vs. the 2 cells shown here; cf. Fig. 6.3) of the A/P line. These border (“B”) cells turn on decapentaplegic (dpp) in response to Hedgehog (Hh) diffusing from the P region, and they join into chains [4848]. No P or A cell can enter the B zone unless it turns on dpp. A-type cells can do so when they approach the A/P boundary, but P cells cannot because en inhibits dpp. Hence, the B/P line restricts cell lineage, while the A/B line does not. If a B clone (shaded) stops transducing Hh due to smo null (smoothened null ), then it forfeits its B identity but not its en-off state and so remains A-type. The B zone then shifts anteriorly because (1) smo null suppresses the Hh receptor Ptc (Patched), and (2) Hh diffuses farther over cells having less Ptc (cf. Fig. 6.6i) [754, 755]. Interestingly, homotypic mingling (which both models presume) occurs when fast-growing clones cross the body midline from one foreleg disc to the other (A-to-A or P-to-P) after they fuse (not shown) [4076]. Segregation of cell types might not actually use qualitatively different linkers (as depicted here) but could instead rely on different linker densities [4070−4074]. Indeed, studies of ptcGOF clones suggest that A and P afﬁnities differ quantitatively because these partly deaf clones behave as if they have an intermediate afﬁnity [2993]. Other molecules (not shown) could maintain epithelial integrity by nonspeciﬁc (or extracellular matrix) binding [1957, 3937]. Adapted from [942].

CHAPTER SIX. THE WING DISC

because the dpp-on state cannot persist for long in cells that are deaf to Hh. 3. Rounding and vesiculation of xenotopic clones. members who ﬁnd themselves outside the walls of their fraternity (the dpp-on stripe) may still attempt to bind together as a satellite group. Ectopic identity is bestowed on A cells around enGOF clones and on P cells just inside the edge of enLOF clones (Fig. 6.6). Both types of clones would “round up” if dppon cells adhere strongly, and further constriction of their dpp-on annuli could lead to invagination, evagination, or detachment as a vesicle, although some of the puckering might arise from growth stimulation due to locally increased Dpp. (Reports do not state whether A-type enLOF vesicles in P areas are totally detached [2447, 2927].) According to this rationale, the rear edge of the dppon stripe restricts mixing of A and P cells because (1) A cells at the edge cannot turn off dpp (Hh signal is too high), and (2) P cells cannot turn on dpp (their en-on state precludes it). The capacity of dpp-on cells to build a “wall” is obvious in mosaics where a dppGOF clone partly restores wing formation in dppLOF ﬂies that lack wings [1839]. Each rescued “winglet” bears a single median clone that is straight and narrow, as if the dpp-on cells were organizing a stripe de novo by aligning and adhering tightly [4848]. When winglets consist of purely A or P tissue, they have A*A or P*P symmetry (where * is the clone). In one P*P case, a dppGOF clone (Fig. 6D of [4848]) was hundreds of cells long (D and V sides) but only ∼5 cells wide. Remarkably, such dppGOF clones create a straight dpp-on ribbon without any guidance from an A/P line or Hh asymmetry, and similar “magic” is seen in dpp+ wings where the dpp-on zone is pushed far into the A region by a smo null clone (Fig. 6.6i) [786]. Evidently, the auto-afﬁnity is polarized along the axis of each cell so that dpp-on cells align rather than clump (i.e., form a band vs. a biscuit). Polarized aggregation in other systems (e.g., ﬁngerprints [1159, 1606] and slime molds [1492, 3594, 4783]) may offer clues to this process in ﬂies. Suspiciously, dpp-on cells are often found where tissues of the same type come together – e.g., tracheal branches [4082], dorsal closure in the embryo [1510, 1515, 3137, 3592, 4854], and midline fusion of discs during metamorphosis [34, 2714, 4429, 4853] (cf. pannier [614, 615, 3504]). Dpp may also help to align the eye’s morphogenetic furrow [724, 930, 1616, 4648] (cf. the chain of cells therein and its buckling properties [4364]). At the tarsus-pretarsus border in leg discs, there is a ring of cells that expresses the homophilic ad-

151

hesion molecule Fasciclin II [2287] but fasII is not under dpp control (cf. dpp-on rings in grasshopper legs [2069]). Rounding of enGOF A clones and enLOF P clones (Fig. 6.6) could thus be due to an inability of circularized dpp-on stripes to break open and linearize. It is less clear why DC0 LOF and ptc LOF clones (which express dpp in a solid circle instead of an annulus) behave likewise: under certain circumstances, ptc LOF clones can straighten as keenly as dppGOF clones [1837]. Overgrowing clones can deform the A/P boundary but apparently cannot break it [497]. Whether a cell can join the “border guard” may actually depend not on its dpp state but rather on receipt of a saturating dose of Dpp, because rounding is also seen for dpp-off clones whose Dpp pathway is activated by a TkvQ253D receptor [569]. Receipt of an autocrine dpp-like (BMP-4) signal is thought to similarly maintain the border between neural and non-neural ectoderm in chick neurulation [4135]. Strangely, cells that both emit and receive Dpp at high doses grow slowly: (1) the endogenous dpp-on stripe seems to slow its mitotic rate during 3rd instar [543, 3813], and (2) cells whose dpp transcription is cranked far above the normal range appear to stop dividing [2457]. Quiescence of this sort should limit the shedding of border cells into the dpp-off A population. Nevertheless, departures must be common because most of the cells in the mature A compartment have expressed dpp at an earlier time in wing disc development [4575]. The chain-forming ability of border cells might be expected to inﬂuence cell morphology. Cells at the wildtype A/P line are aligned and do adopt odd (rectangular vs. polygonal) shapes, but these features only extend one cell diameter on either side and thus fail to reach the dpp-on stripe in late-3rd-instar discs [350]. Conceivably, some combination of Dpp with other inputs ﬁnetunes this line in wild-type ﬂies. Hh can affect adhesivity directly in tergites [2437], and en may also participate because certain combinations of enLOF alleles transform the wing’s A/P border stripe into haltere tissue [1138, 1635]. Regardless of the gadgetry, the existence of some device for aligning the dpp-on stripe makes sense because the precision of all subsequent patterning rests on this slender reference axis [942]. The quandary of A/P afﬁnities was reinvestigated in 2000 by Dahmann and Basler [943]. They found rounding of hh GOF smo null A clones and ci GOF P clones – both of which are consistent with the Border Guard Model because the dpp-on rings that they induce (outside or inside the clone, respectively; cf. Fig. 6.6b) could cinch the clone into a circle. However, rounding was

152

IMAGINAL DISCS

a

Cd

Wild-type

1

2

Cm Cd Te Cp

AC AC

AC

Al

3 4

c

inv

en- OFF dpp- ON en- ON

Al 5

m

GOF

en- ON

b

m

5

en- OFF 1

en-Gal4:UAS-en

4

2

d

3

3 3?

4

P/P

A/A

5

m

2?

2

ptc (en- ON )

3

1

1

2

1

2

3 3

1

?

3

null ptc null en null inv

1

null

2 1 1

2

e

3

2

f

3

4

5

m

1

4

Tethered Hh

3

g

5

m

2

1

h

4

3 4 5

null en null inv

m m

m

5

1

1

3 2 1

2

m 3

4

2

i

5

2

null smo

CHAPTER SIX. THE WING DISC

also seen with ci null hh null A clones and en null inv null ci null P clones – neither of which has a dpp-on ring. Through a series of LOF-GOF tests, the “antisocial” behavior of such “deaf-mute” clones was traced to their lack of Ci and En, rather than the deaf-mute condition per se. Clones lacking Ci and En (ci null A clones, ci null en null A or P clones, etc.) shun both A and P cells and straddle the A/P border. Thus, another type of afﬁnity must exist apart from the intraborder type [364]. We do not know what cell-surface molecules enforce either sort of afﬁnity. E-cadherin is unlikely because dE-cadherin null clones respect the A/P line, although null or GOF dE– cadherin clones do round up and GOF clones from opposite sides of the A/P line can fuse [943]. In sum, the available data favor the Border Guard Model but also argue for other tricks. If cells outside the

153

dpp-on stripe have a common afﬁnity that transcends their A or P afﬁliation, then decreases in this stickiness might explain the old “Cell Competition Conundrum” [4884] – namely, why do slow-growing clones persist at A/P and D/V borders but not elsewhere [1639, 2132, 3947, 3948]. To wit, if the mitotic rates of cells were to alter their adhesivity, then retarded clones could be pushed to the edges of compartments, and those that fail to reach an edge might be squeezed out of the epithelium altogether.

Intercalation is due to a tendency of Dpp gradients to rise Given our current picture of the circuitry (Fig. 6.4), it is now possible to devise plausible etiologies for how venation goes awry in various mosaic wings (Fig. 6.6). In the following cases, the wing’s A-P pattern is formulated as

FIGURE 6.6. Weird wings that reveal the logic of A-P patterning. Gene abbreviations: dpp (decapentaplegic), en (engrailed), hh

(hedgehog), inv (invected), ptc (patched), smo (smoothened). All panels show the dorsal side of a right wing (most bristles omitted for clarity), light vs. dark shading denote en-off vs. en-on states, thick hatching (omitted in some panels) means dpp-on, and arrows mark clone limits. a. Wild-type wing. Landmarks: tegula (Te); proximal, medial, and distal costa (Cp, Cm, Cd); veins 1–5; posterior margin (m); alula (Al); and axillary cord (AC). Inset at left shows the base of a 30A-Gal4:UAS-en wing where en is ectopically expressed in the Te-Cm region [1647]. This region has transformed to AC, and a mirror-image copy of Cd has grown out, possibly due to Dpp elicited at the en-on/off interface. Evidently, this interface does not enforce a vein 3/4 identity immediately, though the dpp-on cells may “bootstrap” themselves to such a level eventually if they have sufﬁcient time to do so (e.g., see g). b. How en controls dpp. Cells whose en gene is on secrete Hh (not shown), and diffusion of Hh into en-off territory turns on dpp (cf. Fig. 6.4). “Xenotopic” clones that express en in the A region (above) thus acquire a “halo” of dpp-on cells, whereas en-off clones in the P region (below) acquire an inner dpp-on “lining.” Only clones that cross the D/V boundary reorganize pattern or grossly alter growth, and they are actually elongated (e.g., e and f) vs. round [1837]. c, d. Symmetries that arise when inv is misexpressed. c. Phenotype of an invGOF (dominant) mutant that expresses inv throughout the wing, but which still expresses en only in the P region [3935]. Unlike en, inv affects how cells respond to Dpp (i.e., A- vs. P-type competence) but does not affect dpp or its regulators (hh, ptc, ci, etc.). Thus, inv acts downstream of en (cf. Fig. 6.4). P/P wings are also seen when a Gal4 transgene forces en – and hence inv – on in the A part of the blade (not shown) [1647], but in that case vein 1 stays intact. Likewise, fat-head LOF wings manifest a P/P phenotype because fat-head normally represses en in the A compartment [4607]. d. Partial A/A symmetry (akin to en1 [1369, 1837]). Surprisingly, en-Gal4:UAS-en represses en and inv in response to initial excess of En [4229], implying negative autoregulation (En en) [207]. A/A wings are also seen with LOF changes in EGFR signaling: dRaf LOF [207], pointed LOF [3804], Ras1 LOF [207], and vein LOF [3929]. The etiology of the EGFR effects is obscure. e, f. Effects of inducing ptc null (e) vs. ptc null en null inv null (f) clones in the vein 1 area. Disparity in clone breadth (3-3 distance) may depend on activation (e) vs. failure to activate (f) en within the clone, which dictates whether dpp turns on outside or inside (zone in f is conjectural) [4229]. In both cases, the ectopic dpp-on tissue induces a symmetric outgrowth, and the R-L-R handedness obeys Bateson’s rule (cf. Fig. 5.3). Weaker (“1221” vs. “123321”) outgrowths arise with dppGOF clones (not shown) [4848]. g. Effect of an en null inv null clone at the P margin. This A-type clone evidently formed a new dpp-on zone (not shown) at its interface with P-type tissue, and that zone fostered the outgrowth of a whole new A and P compartment. The R-L mirror plane (cf. e, f) illustrates an “edge effect” predicted by Meinhardt [2808]. h. Effect of substituting a membrane-tethered Hh (which cannot diffuse) for the endogenous Hh. The wing is small, and veins 3 and 4 are fused, but other features are relatively normal (cf. Fig. 6.13c). i. Effect of a smo null clone of A-type cells at the A/P border (cf. Fig. 6.5). Hh diffuses farther over smo null cells but is not transduced by them, so Hh only turns on dpp when it reaches competent tissue beyond the clone. Evidently, this journey diminishes Hh’s strength because the amount of Dpp fails to foster (1) normal growth, (2) patterning of the P region, or (3) full “123321” duplication of the A region (cf. e, f). Wings (all at same scale) were traced from pictures in [1647] (inset in a), [3935] (c), [4229] (d–g), [4136] (h), and [754, 786] (i). Schematic (b) is based on [354, 1647, 4136, 4229, 4848]. Another “brainteaser” phenotype (not shown) whose etiology was ﬁgured out using known circuits is dpp-Gal4:UAS-tkv [4251] (their Fig. 6b). How duplications arise in Egfr LOF gro LOF wings (not shown) is unknown [3465].

154

“123/45m” [4848], where numbers are longitudinal veins, “/” is the A/P compartment boundary (inserted at every 3–4 juxtaposition), and “m” is the part of the margin behind vein 5. All structures derived from the clone (* = initial site) are underlined, new structures (clonal and wild-type) are bracketed, and dpp-on zones are denoted by “z.” All the analyzed clones run parallel to the A/P line and cross the D/V boundary. 1. enGOF clones in the A region. When en is turned on between veins 2 and 3 (12*3/45m), the ﬁnal pattern is “12{3/4*4/3}3/45m” or “12{3/*/32}3/45m” [4848]. Evidently, a dpp-on zone arises at each en on/off interface (“2z*” and “*z3”), and the extra Dpp evokes growth inside (vein 4 = P-type response) and outside (vein 3 = A-type response) the clone, leading to a roughly symmetric {3z4*4z3} or {3z*z32} subpattern at the initiation site. 2. hhGOF clones in the A region. When hh is turned on between veins 2 and 3 (12*3/45m), a “12{3/4*4/32}3/ 45m” pattern appears to develop (the authors do not label vein 4 [231]). The etiology is likely the same as for enGOF , except that the clone gets its en-on (Ptype) state indirectly via a “Hh en” link. When hh is turned on in the vein 1 area (1*123/45m), the resulting pattern seems to be “1{23/4*4/321}123/ 45m” [231] (but vein 4s can be missing [2058]). Although the whole {23/4*4/321} insert is symmetric, growth is asymmetric about each dpp-on zone: for some reason, more growth occurs outside than inside the clone (“23z4” and “4z321”). 3. Clones in the A region that constitutively activate the Hh pathway (cf. Ch. 5). If an A-type cell “thinks” that it “hears” Hh, then it should behave like a hhGOF clone – i.e., turn en on (Hh en) and dpp off (En dpp). However, as discussed above, this response requires a stronger signal than to turn on dpp (Hh dpp). The threshold for turning en on is reached by ptc null clones but not by weaker ptc LOF clones [2834, 3177, 4136, 4229]. The greater width of ptc null (vs. ptc null en null ) clones in the vein 1 area (Fig. 6.6) [4229] may thus reﬂect an attempt to form a whole P compartment (vs. a dpp-on stripe), but ptc null clones also express dpp unevenly [642] and lack vein 4s (“1{23*321}123/45m”) just like weak ptc LOF clones [754, 3177]. Evidently, cells can cross the “Hh en” threshold but fail to turn dpp off until they make enough En [998]. Indeed, DC0 null clones express en weakly [3177] but create the same “4-less” pattern as ptc LOF clones [2491, 2533, 3177, 4538] via Dpp from

IMAGINAL DISCS

the clone itself (vs. ﬂanking cells) because DC0 null dpp null clones do not cause duplications [2533, 3238]. Only when DC0 null clones are also mutant for Su(fu) do they fully turn en on [3177] (cf. cos2 LOF [3976]). Other components exhibit odd defects: slimb LOF clones participate in making vein 3 and have a small 3*3 span [4538], and a vein 3 arises in the middle of fu LOF ptc LOF (but not fu LOF DC0 LOF ) clones [3177]. 4. dppGOF clones in the A region. Clones whose dpp gene is switched on should make a “1{23*32}123/45m” pattern [4229], but when a weak (Tubulinα1) promoter was used with a dpp transgene, the maximal effect was “1{2*21}123/45m” [4848]. Posterior clones (123/45m*m) also failed to make vein 3 (maximum = “123/45m{m54*45m}m”) [4848], and so did winglets created via dppGOF “rescuer” clones in dppLOF discs (“1*1” or “m*m”) [4848]. This limitation makes sense given that vein 3 depends on Hh directly, rather than on Dpp (cf. Fig. 6.13) [2992]. Ectopic dpp-on clones that employ the T ubulinα1 promoter turn on omb (low threshold) but not spalt (high threshold) [3074]. The latter case is especially instructive. If dppGOF clones do not induce vein 3 (or spalt) when the dpp gene functions at submaximal capacity, then perhaps the dpp-on condition does not impose any positional identity whatsoever. In other words, a cell does not think “If my dpp gene is on, then I must be a 3–4 intervein cell” [4229]. Rather, a cell whose dpp gene is turned on may adopt the A-P level of its environs and then try to “climb” to the 3–4 intervein peak. If its climb is hampered (e.g., by a weak dpp promoter) or aborted (e.g., by insufﬁcient time), then its Dpp level – and hence the “vein state” of nearby wing cells – will never reach the “3–4” state goal. This “Climbing Scenario” contrasts with the “Intercalation Scenario” of the old Polar Coordinate Model because it does not rely on discontinuities per se to stimulate growth. Indeed, the ﬁnal nail in the PC Model’s cofﬁn may be a “123*21/m” small-wing phenotype that arises when smo null A clones (123*/45m) displace the dpp-on stripe (Fig. 6.6i) [754]. According to the Rule of Shortest Intercalation, the 1/m disparity should elicit growth [354], but it does not. Rather, the new growth (*21) occurs where there was no discontinuity – at the original 3/4 site. Subsequent changes in identities also make no sense in terms of polar coordinates but are explicable by the Climbing Scenario. The etiology seems to be as follows [754]. When A cells at the A/P border become

CHAPTER SIX. THE WING DISC

smo null , they can no longer transduce Hh but remain A-type because their en gene is still off. As their Hh pathway shuts down, ptc stops being upregulated and the density of superﬁcial Ptc proteins subsides. Removal of these Hh-binding sites lets Hh diffuse farther. Only when Hh reaches competent smo+ cells can it induce dpp expression, so the dpp-on stripe shifts ahead of the smo null clone (Fig. 6.5) and continues to migrate anteriorly as the clone grows. As the gap widens between the Dpp source and the P compartment, Dpp’s mitogenic stimulation of P-type cells dwindles, so their growth abates. Moreover, the falling levels of Dpp reaching the P region cannot sustain the formerly high A-P positional identities, so “vein states” ebb from “4” to “4–5 intervein” to “5” and ﬁnally to “5–m”. (These identity shifts preclude a “Ratchet Scenario” [3225, 3999] because a ratchet should prevent such demotions [1657].) Within the A region, the smo null clone’s proximity to the dpp-on stripe nurtures its growth until it spans two interveins [754] (wild-type clones are not usually so big [521, 1545]), but here too demotions are evident: cells within the dpp-on stripe sink from a “3–4 intervein” identity to a “vein 3” state (again precluding a ratchet). The probable reason is that Hh’s journey over the smo null territory diminishes its strength so it induces a reduced level of dpp expression. A similar apposition of opposite cell states (≈ 1/m) is seen in a Gal4 line where UAS-en is expressed in a tiny part of the anterior wing (Fig. 6.6a). The full series of elements around a wild-type wing from A to P is tegula (Te); proximal, medial, and distal costa (Cp, Cm, Cd); 123/45m; allula (Al); and axillary cord (AC). When en is turned on in the Te-Cp-Cm region, those elements are replaced by AC (a P-type response to the Dpp gradient), and Cd duplicates in mirror image to yield “AC{Cd}Cd123/45mAlAC” [1647]. Apparently, the abutting of en-on (AC) with en-off (Cd) cells creates a dpp-on zone whose output is too small (or whose “climb” is too brief) to raise cells above a Cd identity. Larger outgrowths (with vein-1 identity) can arise here when en expression is evoked by groucho LOF [998] or polyhomeotic LOF [2728, 3512, 3753]. This climbing or “bootstrapping” tendency of Dpp gradients may be due to a simple law of mass action. To wit, “the range of signaling depends on the amount of signal generated, which in turn depends on the number of Dpp-secreting cells” [3074]. As the tissue around a Dpp source grows, so too does the source. The more it grows, the higher the Dpp concentration will rise, and the more thresholds for gene activation (omb, spalt) and vein induction (1, 2, 3) will be crossed [2457,

155

3225].

At some point, the growth must become selflimiting because discs stop growing even when given extra time (cf. Ch. 4) [542], but the terminal trigger for maturation is obscure [977, 2776, 4886] (cf. servomechanisms [40, 4725]).

The variable height of Dpp gradients makes them appear seamless One of the appealing features of the Polar Coordinate Model was its seamlessness. No singularities were supposed to interrupt the smooth sequence of coordinates around a disc. Even the “12/0” line in the standard clockface depiction of the model was assumed to be invisible to the cells themselves because they were somehow able to interpret “12” and “0” as the same coordinate [1303, 1807]. Only a seamless mechanism, it was thought, could explain why the plane of symmetry in duplicated patterns can be situated anywhere along an organ’s axes, and seamlessness was thought to be antithetical to cell-lineage compartments. There seems to be no restriction on the possible positions or directions of symmetry lines in duplicated patterns. For example, Bryant [522, 524, 525] showed clear cases where the symmetry line divided organs such as sensilla groups, axillary sclerites, tegula, etc. This indicates that the pattern regulates as a whole and that there is no subdivision of the patternforming field into sub-fields during the process of duplication. [526]

In duplicated patterns . . . we found that the line of symmetry could pass through various recognizable structures such as axillary sclerites, notal wing processes, tegula, etc. In these cases, then, only part of the structure is present but that part is duplicated. . . . We conclude from cases such as these that the wing disc is not subdivided into separate morphogenetic fields corresponding to these components of the adult structure. The developmental behavior of cells during regeneration and duplication is defined by their relation to the overall pattern and not to any subdivision of it. [525] The plane of symmetry forms without regard to the boundaries of the named recognizable structures. Furthermore . . . the demarcation between doubled and deficient areas on the disc is imposed without regard to the compartments described by Garcia-Bellido et al. [1376]. Evidently the plane of symmetry recognizes no natural barriers from the center of the disc to the periphery. It is somewhat surprising that sometimes the plane of symmetry goes through a single cell. For example, Fig. 7 shows the plane of symmetry going through the anterior post-alar bristle . . . and Fig. 17 illustrates the plane of symmetry going through a single sensillum. Unfortunately, both of these single cell structures are symmetrical even in normal flies, and so it cannot be proved that in these pattern alterations the symmetry of half a cell is reversed. [3440]

156

An example of this phenomenon is the ability of left and right 1st-leg discs to fuse together to varying extents when cells are randomly killed. The precise mirror image patterns in fused prothoracic legs show either that exactly the same number of cells die in precisely the same location in each leg disc, or that pattern regulation in the left and right leg discs is coordinated to provide similar patterns after different degrees of cell death. [3442]

Interestingly, a similar spectrum of fusion phenotypes can be produced by adjusting the amount of ectopic Dpp that is expressed in the Wg sectors of 1st-leg discs [2954]. Clearly, the dpp-on stripe can operate like a rheostat (vs. a digital switch) [1807]. Ironically, the earliest model for disc regeneration – the 1971 Gradient of Developmental Capacity Model (cf. Ch. 4) – resembles our current view about Dpp’s role [522]. Where did the 1976 PC Model go wrong? Its most obvious mistake was discussed in Chapter 5 – namely, its premise that regeneration obeys the rules of normal development. In fact, repatterning in the foreleg disc proceeds via an abnormal contact between the peripodial and columnar layers (cf. Fig. 5.10c) [1472]. The ability of the upper medial quadrant to reform a whole P compartment after being “infected” with only a few en-on cells must be due to its dpp-on halo, which fosters the growth of the colony during the week needed for full regeneration [3808] (≈ a self-feeding Minotaur). In a similar way, perhaps, en null inv null clones in the P compartment instigate colonies of A-type cells that can grow to the size of a whole A compartment (Fig. 6.6g) [4229]. Whether the ectopic dpp-on stripes (that arise at each interface between en null inv null and wild-type tissue) climb to a 3–4 intervein state depends on the time available: late-induced clones never attain a 3–4 level. Moreover, the height of the “valley” between adjacent Dpp peaks depends on the distance between them. For example, a “123/4*5m” clone can yield a “123/4 {4/3*3/4}5m” pattern (valleys = 4–5 intervein at “{” site and 2–3 intervein at “*” site), whereas a “123/45m*m” clone can produce a “123/45m{m54/321*123/45}m” pattern (valleys = m at “{” and 1 at “*”) [4229]. The farther an ectopic “/” peak is from the endogenous peak, the deeper the intervening valley can sink. In these two examples, the valleys sank to “vein 1,” “2–3 intervein,” “4–5 intervein,” or “m”. Evidently, the valley can occupy any level of the gradient, as can the peak [3753]. Mirror planes between subpatterns can reside at valleys or peaks when ectopic dpp-on stripes arise within compartments (e.g., dppGOF A clones [4848]).

IMAGINAL DISCS

This ability of mirror planes to “slide” freely along Dpp gradients reveals a subtler misstep in the history of the PC Model. It was erroneous to assume that compartment boundaries would have ﬁxed cellular fates (e.g., 3–4 intervein) in reconstituted patterns [904]. In fact, Dpp gradients can produce patterns that are as seamless as the patterns that the PC Model can theoretically create by “ironing out” discontinuities. This scaffolding strategy is analogous to hanging clothes (structures) on a clothesline (gradient), where the poles can be raised to any height and the clothesline can be slackened to any depth. No one ever suspected that the scaffold itself could be so amazingly ﬂexible or so utterly invisible. If the PC Model is incorrect, then why do fragments obey the Rule of Shortest Intercalation (cf. Ch. 4)? A ﬁnal answer must await further studies, but a guess may be attempted. Fragments might not “take the long way around” because any new dpp-on zones that they acquire (via wounding [489, 2856] or A/P confrontation [1472]) only raise the gradient levels of nearby cells. For a cell’s identity to “jump” to the opposite side of the fate map (cf. Fig. 6.6a), it would need to switch at least one selector gene, but Dpp does not alter those states. Fragments probably obey the Reciprocity Rule for a similar reason: wound edges are unlikely to replace structures on the opposite side of the disc unless they are assisted by unusual contacts. Indeed, the choice between regeneration and duplication for any given piece is critically dependent on the mode of wound healing [947]. In summary, the basic chain of events along the A-P axis is: En (P region) Hh (diffuses across A/P line) Dpp (diffuses in both directions) target genes. Those targets of Dpp then cooperate with other targets of Hh to establish the pattern of veins (see below).

A Wg gradient specifies cell fates along the wing’s D-V axis In 1996, Wg was shown to act as a morphogen along the wing’s D-V axis [4849] by the same sort of evidence that established Dpp’s morphogen status in the same year [2455, 3074]. Wg’s long-range action was monitored by the target genes vestigial (vg) and Distal-less (Dll) – both of which encode transcription factors [836, 1686, 4681] – and neuralized (neu), a marker for SOPs [417, 2387, 3374]. All three genes are positively regulated: their expression wanes when Wg signaling is suppressed (e.g., in arm LOF clones or in t.s. wg LOF larvae exposed to high temperature [4849]), and they turn on when the Wg pathway is ectopically activated in the wing pouch (e.g., in sgg LOF clones [351, 1040]). Wg’s effect on these target genes (Wg vg, Wg Dll, Wg neu) must be direct (vs. a signal relay mode)

CHAPTER SIX. THE WING DISC

because (1) ectopic expression of a membrane-tethered form of Wg induces vg, Dll, and neu expression only in adjacent cells, and (2) activation of the Wg pathway in ectopic armGOF clones turns on vg, Dll, and neu only within the clone [4849]. Moreover, each gene appears to respond to a different threshold level of Wg (Tneu > T Dll > Tvg ) because (1) ectopic Wg-secreting clones induce neu, Dll-lacZ, or vg expression in nested circles (radii: rneu < r Dll < rvg ) when a strong promoter drives wg; and (2) neu fails to be expressed with a weaker promoter [4849]. As expected for a gradient mechanism, the seriation of the nested circles matches the endogenous rainbow of bands that straddle the wg-on stripe at the D/V compartment boundary (Figs. 6.2 and 6.3), although the punctate neu transcription is excluded from wg-on cells.

Perpendicular (Dpp × × Wg) gradients suggest Cartesian coordinates In 1997, the above results (from Zecca and Basler in ¨ Zurich and Struhl in New York) were corroborated by Neumann and Cohen in Heidelberg [3087, 3091]. One new insight came from using dpp-Gal4:UAS-wg to lengthen the point of overlap between wg-on and dpp-on domains into a solid A/P stripe. Dpp and Wg do not antagonize one another in the wing as they do in the leg [1833, 4277], so this sort of coexpression is stable. Such discs can double in length via expansion of the pouch [2252, 3091] or duplicate via inception of an extra pouch [2252]. This synergistic hyperplasia agrees with the commonly observed fact that dpp-activating clones must cross the (wg-on) margin to cause ectopic outgrowths [1839, 4848]. 1. Dpp pathway: dppGOF [4848] and brk LOF [619] clones only cause outgrowths when they cross the margin. 2. Hh pathway: Outgrowths (123y321 where y is the clone) arise around the following anterior clones when they cross the margin: hhGOF [231, 4848], ptc LOF [754, 3077, 3177], cos2 LOF [3976], slimb LOF [2060, 4279, 4538], and DC0 LOF [2491, 2533, 3177, 3238, 4538], although slimb LOF and DC0 LOF cells have identities closer to vein 3 [2058, 3238, 4538]. 3. Engrailed on/off boundaries: Doubly mutant en null inv null clones generally must reach the distal or posterior wing margin to reorganize the wing’s vein pattern [4229]. This result conﬁrms older studies using weaker alleles: en1 [2441] and en LOF [2447] clones incite outgrowths only when they reach the P margin. The same is true for enGOF clones in the A compartment [4848]. The ultimate proof of this “Dpp-Wg

157

Intersection Rule” for outgrowth is a Gal4 line that expresses en throughout the anterior wing pouch except the margin [1647]: the A region adopts a P pattern (veins 2 and 3 transform into veins 5 and 4, with an ectopic posterior cross-vein), but no outgrowths arise. Because an intersection (or at least proximity) of dpp- and wg-expressing domains seems essential for wing outgrowth (natural or ectopic) [164, 643, 1976], wing outgrowth appears to obey the same “Dpp + Wg” rule that has been proposed for leg outgrowth [620, 3862]. The spot where the endemic dpp-on and wg-on stripes meet could be a reference point for positional information [1427]. Indeed, the vg gene’s “quadrant” enhancer is ﬁrst activated there [2219, 2254], and this spot is unique in how it responds to Notch [2462] and Dpp levels. Suppression of Dpp pathway genes causes apoptosis and nicking there [2455], whereas excessive Dpp signaling causes apoptosis elsewhere along the margin [12]. An extra “Dpp + Wg” spot appears to arise in the posterior margin of twins LOF wing discs [4418] and in the anterior margin of lethal(2)giant discs LOF wing discs [545, 558, 559, 2976]. The result in each case is a wing duplication. Twins is a regulatory subunit of protein phosphatase PP2A that is involved in both the Dpp and Wg signaling pathways (Fig. 5.6), so LOF mutations could be short-circuiting the system somehow. The etiology for l(2)gd LOF is obscure. The “world record” for multiple winglets from a single disc is 8: the extra 7 were induced by coexpressing large amounts of Dpp and Wg along the D/V boundary [2254]. All these clues are consistent with a “Dpp + Wg” trigger. However, as in the case of the leg disc (cf. Ch. 5), the trigger for wing outgrowth seems to only depend on Wg indirectly, if at all [1074, 4682]. The formula may be “Dpp + X = distalize!,” with factor “X” being elicited by circuit elements at the margin [2092] that happen to overlap the wg-on band. The evidence against a direct role for Wg includes the following (see [2092, 2252] for more): 1. Wing growth proceeds virtually normally when wg function is repressed throughout the disc in 3rd instar via a t.s. wg allele [880] or when it is repressed at the tip via dpp-Gal4:UAS-scabrous [2462]. 2. DC0 null wg null double-mutant clones induce partial duplications of the blade as readily as DC0 null (margin-crossing) clones [2533, 3238], implying that Wg is dispensable for ectopic tip formation. 3. sgg null clones (whose Wg pathway is activated) fail to induce outgrowths [1040, 2252].

158

4. wgGOF cannot rescue wing growth in discs that are stunted due to apnull or Ser null or Su(H)null or vg null [2092, 2252], but stunted wg LOF discs (as well as ap null , Ser null , or Su(H) null discs) can be rescued by vg GOF [2219, 2252]. If the “Dpp + Wg” rule is wrong, then how can duplications in dpp-Gal4:UAS-wg discs be explained? In those cases, the ectopic pouch always arises where the dpp-on-wg-on stripe meets a native vg-on spot in the notum [2252], so the formula may actually be “Dpp + Vg + X = distalize!” where “Wg X” to provide the missing ingredient [2254]. In the absence of Vg, the main effect of excess Wg is overgrowth of the hinge (vs. the blade per se), with negligible impact on patterning [459, 2252, 2254, 3088]. This ability of wgGOF to uncouple growth from patterning is undoubtedly relevant to the mystery of how Dpp and Wg gradients foster uniform (vs. concentration-dependent) growth throughout the epithelium (cf. Ch. 4) [3862], but its exact implications are unclear.

But cells do not seem to record positional values per se Are the perpendicular Dpp and Wg stripes (Fig. 6.3) really reference axes in a bona ﬁde Cartesian coordinate system (cf. Fig. 4.3b) [4682]? In theory, each cell could pinpoint its location by (1) deducing its quadrant from the states of its selector genes (en and apterous, see below), and (2) ascertaining its site within the quadrant by measuring the amounts of Dpp and Wg. However, there is no evidence that wing disc cells record their positions with any such precision. Rather, they switch on only a few target genes per morphogen, based on preset threshold levels. The disc is thus subdivided into a checkerboard of districts (≈ superimposed French Flags). Cells would know their district (as a binary address of on/off “memory genes” omb, spalt, vg, Dll) but would not remember their exact location. The same conclusion was reached for how embryonic cells “interpret” the Bicoid gradient (cf. Ch. 4) – viz., as a code (of gap, pair-rule, and segment polarity gene states) whose resolution never attains the graininess of individual cells. The upshot of this line of reasoning is that the PI Hypothesis is only partly correct here. Morphogen gradients do inform cells of their positions, but cells do not record “positional values” per se. Without any quantitative positional values, there is no need for an “interpretation” step. Cells go directly from gradients to a few qualitative states, thus bypassing a positional-value stage. These states are then used as anchors for pattern elements (e.g., veins, see below) and function more like

IMAGINAL DISCS

prepattern singularities. Overall, the mechanism would be a PI-prepattern hybrid [3087].

Wg’s repression of Dfz2 is inconsequential In 1998, through various LOF and GOF experiments, Cadigan et al. showed that Wg reduces the amount of Dfz2 (its receptor) [595]. They also found that higher densities of Dfz2 retard Wg degradation and sensitize cells to Wg signal. Based on these facts, they proposed a “gradient shaping” scheme like the Rock Concert Scenario discussed above for Dpp. The Wg gradient is steep near its source and shallow elsewhere, but nearby cells may sense a lesser slope due to fewer receptors (Wg Dfz2), while outlying cells may sense a greater slope due to more receptors. The biphasic (spire-ramp) proﬁle could hence be perceived as a smooth gradient with constant signal-resolving capacity per unit distance [2457], and the reciprocal proﬁles of Wg and Dfz2 would make sense in terms of the “Wg Dfz2” reciprocity. In 1999, however, this scheme was essentially disproven by the discovery that Dfz2 null wings develop normally [734]. The smoothing idea could not be saved by invoking a redundancy of Dfz2 with Fz [734] because no “Wg fz” link exists: Fz is distributed uniformly in wing discs [3264]. Nor can Dfz3 rescue the hypothesis because it manifests an opposite sort of regulation (Wg Dfz3) [3977]. Hence, whereas the “Wg Dfz2” link clearly operates in a wild-type context (unlike “Dpp tkv”), its ability to “shape” the Wg gradient appears to be essentially inconsequential.

Apterous’s role along the D-V axis resembles Engrailed’s A-P role Because Dpp and Wg have similar diffusion ranges, the evolutionary decision as to which morphogen to use along each axis must have been arbitrary from an engineering standpoint. The only obvious constraint is that a single morphogen cannot be used for both axes unless they are patterned at different times [4724]. The same rationale applies to the compartments whose boundaries evoke Dpp and Wg. It turns out that separate selector genes (also arbitrary) are used in overlapping areas to delineate four quadrants. Until 1993, Garc´ıa-Bellido’s Selector Gene Hypothesis (cf. Ch. 4) [1358] rested mainly on a single gene (engrailed) and its idiosyncratic phenomenology. In that year, a similar suite of properties was found for a second gene (apterous) [361, 2445], thus bolstering the idea [354]. 1. Like engrailed, apterous (ap) encodes a homeodomain transcription factor [290], but Ap also has two LIM (protein docking) domains [418, 826, 3159, 3601]. The

CHAPTER SIX. THE WING DISC

159

b V

a

c

ap- ON

ap- OFF

vTR

wg- ON

V P

A

D

D

TR

dTR

ap- ON

Key: CS MS

ap- OFF TR

e

2 3

st al

4

P

A P

3rd in Engra star iled

D di

oxima pr

l A

DR

d

mTR

PR

GOF

Dlw

D/D

f DlwLOF V/V

dTR' mTR' mTR dTR vTR vTR'

FIGURE 6.7. The role of apterous in establishing the wing margin.

a. Mature right wing disc. Directions (compass at left) are A, anterior; P, posterior; V, ventral; D, dorsal. Prospective D vs. V regions of the disc are indicated (dark vs. light shading), as are the wing pouch (thin dashed line) and future margin (thick dashed line). The D part expresses apterous (ap); the V part does not. The margin expresses wingless (wg), as do the pouch edge and a notal stripe (not shown). The Wg band straddles the D/V boundary (in contrast to the Dpp stripe, which abuts the A/P line; Fig. 6.6a). b. Effects of xenotopic ap clones inferred from adult defects and GOF data. When ap is misexpressed ventrally (above), it turns wg on at the edge of this GOF area [2851]. Based on their ectopic margins (c) [1038], ap LOF clones in the D region (below) must also turn wg on peripherally. c. The normal margin (center) has a triple row (TR) of MS (mechanosensory, straight) and CS (chemosensory, curved) bristles. The medial TR (mTR) has stout bristles, and the dorsal TR (dTR) has CS bristles, while the ventral TR (vTR) has a 4:1 ratio of slender MS to evenly spaced CS bristles. New TRs develop around apGOF tissue in the V region (upper oval) [2851] and ap LOF clones in the D region (lower oval) [1038], whenever these spots are anterior to vein 2. The apGOF V spots have a “dTR-mTR-E-vTR” polarity (E = clone edge) from inside to outside, whereas the ap LOF D clones have opposite polarity [1038]. d. Right adult wing (D surface). Only stout bristles are drawn. Along the front is TR. More posteriorly are a double row (DR) and a posterior row (PR). DR (like TR) has innervated bristles, but PR only has nonsensory hairs [1741, 1837]. The DR/PR transition (at vein 3) regresses to the A/P line (∼ vein 4 [350]) if En expression in the A region (which arises in the 3rd instar [350]) is blocked [1837, 4229]. Conversely, it advances farther anteriorly if Hh is overexpressed [2992]. Why the anterior extent of the en-on area is greater distally than proximally (not shown) is a mystery [350]. e, f. Symmetric (D/D or V/V) phenotypes caused by GOF (e) or LOF (f) alterations at the Dorsal wing (Dlw) locus [4325]. Strangely (via transvection?), the same mutation (Dlw1 ) switches cell fates in opposite ways (V-to-D vs. D-to-V), depending on whether it is in heterozygous (Dlw1 /+ ﬂy = GOF) or homozygous (Dlw1 /Dlw1 clones = LOF) condition. In each case, the effect is fully penetrant but partly expressed (i.e., not all bristles are transformed). V/V wings can also be created by using ap-Gal4 with UAS-Ser and UAS-fringe to restore Notch signaling, a wg-on stripe, and a wing margin (albeit a V/V one) in an ap LOF background [2850, 3158]. Panel a is after [361], b and c are based on [882, 1038, 1741] (although clones are typically more elongated along the proximal-distal axis than shown here [521, 1373, 1545]), d is adapted from [350, 524, 1224, 1741, 1837], and e and f sketch phenotypes from [4325]. See also App. 7.

gene is named for its missing-wing LOF phenotype [470, 3848, 4119, 4692, 4693]. Although ap is expressed in legs and antennae, these structures develop normally in ap null ﬂies [826] so, unlike en, ap appears to function only in certain discs (wing and haltere). Part of the reason may lie in the leg’s lack of D/V lineage restraints [354, 1800, 4076].

2. In the wing disc, Ap is expressed only in dorsal anlagen (notum, dorsal wing and hinge), and the distal edge of the ap-on area aligns with the wing margin (Fig. 6.7) [352, 826, 1038, 4682, 4683]. This congruence implicated ap as a selector gene because the margin is a boundary that somatic clones fail to cross starting in mid-2nd instar [361]. Indeed, that is when

160

ap expression becomes detectable [1074, 2251, 4543, 4683], suggesting that the lineage restriction could be due to ap. At one time, it was thought that the restriction was due instead to a zone of mitotic quiescence [494, 498, 3154]. However, this notion proved false [352], partly because the (wg-dependent) quiescence arises too late to explain the (ap-dependent) lineage segregation [2079, 2846, 3374, 3979, 4683]. 3. As with en, switching the ap-on/off states of cells causes (1) compartmental switches in downstream genes (e.g., integrins) [361, 498, 4118], (2) homeotic changes in adult structures [1038, 3158], and (3) the emergence of boundary identities at on/off interfaces [1038] (Fig. 6.7). The initiation of the ap-on state dorsally depends on Hh (although the geometry makes no sense) [2251], while its off state ventrally is maintained (like that of en anteriorly) by Polycomb [3752, 4327]. 4. Like en LOF clones in the P (en-on) region, ap LOF clones in the D (ap-on) region cross the compartment boundary when near it and round up when far from it [361], and Polycomb LOF (ap-on?) clones cross in the other direction [4327]. Thus, ap could control compartment-speciﬁc afﬁnities (Selector Afﬁnity Model), but it might be acting indirectly (Border Guard Model) [352, 361]. In the absence of Ap, a border still arises but is ragged [2251], possibly due to apoptosis [4683]. The ability of Fringe to rectify this border without any Ap activity rules out the simple Selector Afﬁnity Model (see below) [3158]. The asymmetry of D vs. V bristles at the wing margin (Fig. 6.7) makes it possible to distinguish mirror-symmetric (D/D or V/V) and inverted (V/D) margins from the wild-type (D/V) condition. (See [1368, 3158, 3624, 4189] for vein asymmetry “corrugations.”) When ap null clones arise dorsally (and anterior to vein 2), they induce an ectopic V/D margin at their perimeter, with V-type bristles inside and D-type bristles outside [1038] (cf. similar spots in ap LOF wings [4118]). Because the endogenous margin also arises at an ap-on/off interface, the implication is that ap is responsible for instigating the margin. The ability of ap null clones to autonomously convert cells from D- to V-type further implies that ap also controls regional (D vs. V) identities.

Chip cooperates with Apterous, and ‘‘Dorsal wing’’ acts downstream Two other genes besides ap have been found to cause similar switches in cell identities – Chip [1217] and Dor-

IMAGINAL DISCS

sal wing (Dlw) [4325]. Chip was isolated independently by (1) a genetic screen for agents that bridge enhancers to promoters [2939, 2940], (2) a screen for mutations affecting the wing margin [1217], and (3) a two-hybrid screen for proteins that bind Ap [1217]. LOF-GOF analyses led to some odd results [1217] (cf. a similar paradox for Dl and N [75, 990, 1204]). These riddles are listed below, alongside solutions offered by a 1998 “Ap-Chip Tetramer Model” ´ proposed by Fern´andez-Funez et al. [1217]. “Boxes” allude to graphics in Fig. 6.8b (see [2854, 3600] for further evidence). Riddle

Explanation

1. Chip-LOF clones behave like ap-LOF clones (i.e., D clones induce margins, whereas V clones do not), despite the fact that Chip is on ubiquitously (ap is only on dorsally). The ectopic margins have V/D (inside/outside) polarity, indicating D-to-V transformation. Expression of ap is normal within the clone, so ap is not acting downstream of Chip.

1. Chip-LOF and ap-LOF have similar effects because the tetramer concentration drops when either Chip or Ap is in short supply (boxes 2 and 3). Defects arise when it drops below a threshold for target gene activation. Chip may normally be the limiting factor because Chip (not ap) has a haplo-insufﬁcient (Chip-null/+) effect [118, 1217].

2. Chip-LOF and Chip-GOF have similar effects. Misexpression of Chip in the A/P zone (via dppGal4:UAS-Chip) induces margin bristles at the P (but for some reason not A) edge of this band, and the new margin is conﬁned to the D region, implying a D-to-V switch inside the Chip-GOF band.

2. Misexpression of Chip in the A/P zone has no effect ventrally because Ap is absent and Chip has no independent role. Dorsally, the extra Chip proteins sequester many Ap proteins in inactive trimers (box 4). Hence, the effects of Chip-GOF match those of Chip-LOF – a counterintuitive fact.

3. Overexpression of Chip in the D region (via ap-Gal4:UAS-Chip) suppresses wing development, but overexpressing ap with the same driver causes no such defect (although dorsal ap-GOF can have minor effects [2851]).

3. Excess Chip in the whole D region likewise diverts Ap into trimers (box 4), leading in this case to wing loss. In contrast, excess Ap is relatively harmless because Ap cannot distract Chip into any sort of inert complex (box 5).

4. The wing-loss defect of ap-Gal4:UAS-Chip can be rescued by simultaneously expressing ap in the same domain (apGal4:UAS- Chip:UAS-ap).

4. Jointly raising the doses of Chip and Ap will make more tetramers (box 6), but this analog change may be moot if Ap’s targets respond digitally (on vs. off).

CHAPTER SIX. THE WING DISC

According to this model, which has since been conﬁrmed [2851, 3159, 3600, 4456, 4457], Ap can only activate its target genes as a linear “Ap-(Chip-Chip)-Ap” tetramer. Within the tetramer, a Chip dimer links two Ap proteins. Chip proteins dovetail via a “DD” dimerization domain, while Ap’s LIM domains bind Chip’s “LID” (LIM-interaction) domains. Tetramers may serve a DNA-looping role by forming a “DNA-{Ap-(Chip-Chip)Ap}-DNA” bridge (discussed below). The Ap-Chip gadgetry recalls the AS-C story (cf. Ch. 3) insofar as a patterned agent (Ap ≈ Achaete) must dimerize with a ubiquitous partner (Chip ≈ Daughterless) to activate target genes (cf. Tango [899, 4022, 4548]). Might there exist an antagonistic interloper like Emc which diverts a partner(s) into inert dimers? Indeed, there is. Like Emc, Beadex is a protein with dimerization (LIM ≈ HLH) domains but no DNA-binding (homeo ≈ basic) domain [2854, 3908, 4858]. Consistent with the notion that Beadex is a competitive inhibitor of the Ap-Chip union, Beadex binds Chip (but not Ap) in vitro [2854], BeadexGOF mutations counteract Ap’s activation of target genes in vivo, and Beadex LOF alleles augment Ap’s effectiveness [2854, 3908, 4577]. One amusing twist in the Ap-Chip-Beadex operetta not seen in the Ac-Da-Emc imbroglio is a link that goes beyond the protein level. In early-mid 3rd instar discs, expression of Beadex is conﬁned to the D compartment just like Ap, implying that Ap Beadex, and this link has been veriﬁed (Beadex turns on wherever ap is misexpressed) [2851, 2854]. There are many reasons why genes evoke antagonists [3347, 3399, 3558]. However, in ap’s case the answer is not obvious because ap is a selector gene, and digitally operated genes should not need the kind of analog modulation that ﬁne-tunes the AS-C’s “SOP Computer” (cf. Ch. 3). The reason here appears to be that Ap’s level must be lowered enough to allow installation of new 3rd-instar circuitry (involving Serrate), but not so low that Ap cannot execute its residual selector duties (e.g., D-V adhesion) [2853]. Another lingering mystery concerns the most intriguing of the screens that snagged Chip. Various lines of evidence [274, 1092, 1441, 4204, 4414] – mostly concerning the “insulators” su(Hw) [607, 1401, 2666], scs-scs [2330], and Fab-7-8 [212, 1680, 1681, 2844, 4877] – imply the existence of a “Kilobase Spanner.” This mythical device allows enhancers to control target promoters over large (multikb) distances [2188, 2511, 3002, 3577, 3975], perhaps by knotting the intervening DNA [275, 606, 1092, 2939, 3001]. Morcillo et al. hunted for genes that encode the elusive spanner’s components and reported their ﬁndings in 1996 [2940]. To as-

161

say for spanner-disabling mutations, they used the cisenhancer that turns the cut gene on at the wing margin. This enhancer is 85 kb upstream of the cut promoter [2001] – a chasm almost as wide as the entire AS-C (cf. Fig. 3.4). Chip was one of three genes that they recovered. (The other two are enhancer-binding proteins.) Indeed, Chip was also recovered in a screen for factors that can bridge a 30-kb gap within the AS-C itself (between the DC cis-enhancer and the scute promoter) [3504]. Assuming that Chip is part of the Kilobase Spanner, why should Ap need such a tool? Perhaps the cis-enhancers that Ap binds (to control its target genes) are too far from their cognate promoters to be effective without help from Chip. At the Ser locus, for example, this span (∼10 kb) might be big enough to pose such a handicap [163, 164]. Flies that are heterozygous for a LOF allele of Dorsal wing (Dlw LOF /+) have D/D wings, and V/V patterns are induced by homozygous Dlw LOF /Dlw LOF clones, although both kinds of transformation are incomplete [4325]. Because the latter clones do not evoke margins at their borders, Dlw must be acting downstream of ap and Chip (in a nonsignaling pathway) to implement D-vs.V identities. Thus, Dlw’s D-V role resembles invected’s role downstream of en along the A-P axis. In both cases, parallel pathways (diverging from en or ap) implement cell identity vs. trans-border signaling (cf. Figs. 6.4 and 6.8) [2448].

Serrate and Delta prod Notch to evoke Wg at the D/V line Signaling between the D and V compartments relies on the Notch pathway, which mediates Ap’s control of Wg (Fig. 6.8). The key players are as follows: Wingless (Wg) Notch (N) Serrate (Ser) Delta (Dl) Fringe (Fng) Cut

Morphogen for the wing’s D-V axis. Slave of Notch. Activator for Wingless and Cut. Receptor for Delta and Serrate. Dorsal ligand for Notch at the D/V line. Slave of Apterous. Ventral ligand for Notch at the D/V line. Slave of unknown masters. Muzzler of Serrate. Creator of boundaries. Slave of Apterous. Patron of Wingless. Killer of Delta and Serrate. Slave of Notch.

Wg’s targets include achaete and scute [2079, 2839, 3374, These proneural genes elicit bristles on either side

3690].

162

IMAGINAL DISCS

?

Ap Chip

A p

Ap Chip

? NDl

5

Ap Chip

Ap Chip

Fng

V-type

3 Ap

Ap Chip

Ap Chip

Ap Chip

Ser

Chip

Ap Chip

Dlw

4 Chip

"AND" GOF Ap 6 Ap Chip Chip

p A

D-type

"OR" GOF

Ap Chip

NSer Wg

2

Ser

and

"OR" LOF

Chip

b1

Ap Chip

wildtype

Ap Chip

a

c Dl

D

V

A

Dl

V

D P

d ap wg ac

D V

PNS

PNS

SOP

Ap

f 1 1 0

D-type

1 0

1

?

0

0 0

0 and

1

Wg

0

0

1

(mute)

Wg

0

1

(deaf)

1

and 1

(deaf)

1

(mute)

e

1 0 1

?

V-type

CHAPTER SIX. THE WING DISC

of the wg-on stripe (Fig. 6.2) [882, 1741, 1851]. The wg-on stripe spans the whole margin, but the ﬂanking Ac-Sc stripes only arise in the A half [912, 2079, 3982], perhaps because En represses them in the P half (En AS-C) [882]. The two Ac-Sc stripes express Dl and Ser strongly, while the center wg-on stripe expresses N strongly [988, 1951]. This alternation of high-Dl with high-N resembles the notum [3270, 4428], where the stripes are also ∼4 cells wide (cf. Fig. 3.8) [2079]. Marginal SOPs typically emerge at the inner edge of each high-Dl stripe (Fig. 6.8) [352, 882, 1851, 3689]. This trend suggests that Wg imposes a small gradient of proneural potential within the ﬂanking stripes [356, 882]. Interestingly, when cells at the interface are prevented from transducing Wg (by dsh LOF or arm LOF clones), then cells farther out can become SOPs [882]. This result recalls the

163

ac LOF mosaics whose bristle shifts led Stern to postulate proneural clusters in the ﬁrst place (cf. Fig. 3.3) [4095, 4096]. Most cells in the middle (wg-on) stripe express cut [352, 353, 373, 882], and cut LOF discs have a single broad DlSer (Ac-Sc?) stripe instead of two thin ones [882, 988]. This phenotype recalls the broad Ac-Sc stripes on hairy LOF legs (cf. Fig. 3.9) [3193] and suggests that Cut’s role is to bisect the margin’s Ac-Sc stripe, just as Hairy subdivides the leg’s Ac-Sc stripes. Cut could “carve” the gap by repressing the AS-C (or Dl and Ser) as a transcription factor [370]. An antineural role for cut would explain why wg GOF [459, 2252, 3088], sgg LOF [351, 2252, 2839, 3958], and dshGOF (to a lesser extent) [151] cause conﬂuent lawns of bristles with no cut-like bald areas inside them: such clones fail to turn on cut, which is directly under N – not wg – control

FIGURE 6.8. Circuitry that enforces D vs. V identities of wing cells and turns wg on at the D/V line.

a. Generic wing disc cell (cf. Fig. 2.7 for icons) showing the main control circuit for Wg output. Abbreviations: Ap (Apterous), Dl (Delta), Dlw (Dorsal wing), Fng (Fringe), N (Notch), Ser (Serrate), Wg (Wingless). NSer and NDl are hypothetical Fngmodulated forms of Notch that are receptive to Ser or Dl [990, 1250]. Fng’s damping of the Ser-N trigger has been well documented in vivo [1250, 3246], as has its facilitation of Dl-N binding in vitro [510]. The circuit works as follows. If ap is on (e), then the cell expresses Ser and Fng and adopts D-type identity (via Dlw) but cannot receive Ser signals because Fng blocks the Ser-N interaction. If ap is off (f), then the cell expresses Dl (Dl’s upstream controls are uncertain [2253]) and adopts V-type identity. Dlw (under Ap control) should allow D vs. V cells to interpret symmetric Wg gradients asymmetrically. The circuit allows two types of discourse: Ser-signal “speakers” with NSer “listeners” or Dl-signal “speakers” with NDl “listeners.” Both dialogs should turn wg on. Validation of the circuit’s logic comes from (1) heating t.s. N LOF larvae, which erases wg and cut expression in border cells [2839, 3689], and (2) expressing Ser or fng in the D region of ap LOF discs, which restores Notch signaling and a wg-on stripe but not D identity (hence creating V/V symmetric wings) [2850, 3158]. b. Stoichiometry of Ap-Chip interactions and etiology of abnormal traits. Wings look wild-type when Ap and Chip are equimolar (boxes 1 and 6) or when Ap is in excess (box 5), but not otherwise (X’d boxes). Ap binds DNA (not shown) but can only activate target genes when it forms a tetramer (oval) with Chip (box 1). The tetramer is thought to be a Chip dimer with an Ap at each end. Reducing the amount of Ap (box 2) or Chip (box 3) lowers the number of functional complexes. Excess Chip (box 4) diverts Ap into inert trimers, whereas excess Ap (box 5) has no such effect. Raising the dose of both partners (“AND” GOF) maintains the balance and makes excess tetramers (box 6 shows a doubling). The counterintuitive LOF-GOF genetic data only make sense in terms of this sort of jigsaw-puzzle logic. See text for further details. Oddly, Ap and Chip show no such interdependence in leg discs [3474]. c. Mature right wing disc. Shading denotes D (dark) vs. V (light) compartments, and dashed line delimits the pouch. d. Enlarged part of the anterior D/V border (box in c). Hexagons are cells. All cells in the D region express Ap. Zigzag lines delineate 3 zones (horizontal bars above): a median stripe where wg is on (also cut and N: not shown) and ﬂanking “proneural stripes” (PNS) where achaete (ac) is on (also scute, with intense Dl and Ser but meager N: not shown) [988]. Sensory organ precursors (SOPs, black cells) arise in each PNS along the medial (high-Wg) edge [3689]. In cut LOF discs, the wg-on cells express ac [882, 988], so Cut’s normal role must be to split the otherwise broad ac-on stripe into two parts – the same sort of subtractive logic that is used in the notum (cf. Fig. 3.8f ). e, f. Schematics of cells ﬂanking the D/V line. The V cell is drawn as if ﬂipped around to face the D cell. States of variables (cf. a) are recorded as “1” (present and active) or “0” (absent or inactive). Black circles indicate determining factors. “Mute” and “deaf” denote inability to send or receive particular signals (Ser above, Dl below). The functioning of the circuit is explained under a above. The net result is that both cells are induced to secrete Wg. Circuitry in a is compiled from [1040, 2216, 2252, 3089, 3689] in general and [1217] for Chip, [1991, 1992, 2253] Fringe, and [4325] Dlw. Panel b illustrates data from [1217, 2851, 4456]. It is not known whether the tetramer’s symmetry is rotational (as shown) or reﬂective. Panel d is adapted from [882, 2079, 3689, 3690, 3979]; and the dialog illustration (e, f) is based on [988, 994, 1074, 2839, 3246]. N.B.: Input/output ports (raised rectangles) on the cell surface are drawn at arbitrary apical-basal levels (apex laced with microvilli). The reciprocity of the D/V dialog is a vivid realization of Boundary Model II (cf. Fig. 5.4a) [2808], whereas signaling is unidirectional across the A/P line (cf. Fig. 6.4). Evolution might have opted for symmetric signaling here because the wing’s D and V surfaces must match so precisely.

164

[353, 2839, 3089]. Similar reasoning might explain bristle densities in sgg LOF clones on the legs [1039, 1833, 4666], although

the cut-like agent at the leg’s V midline is unknown. In each case, Wg would foster a prepattern (i.e., 1 or 2 proneural stripes) [3374]. Thus, wg qualiﬁes as the kind of prepattern gene sought by Stern (cf. Ch. 3) [886, 4095], while Wg’s ability to bias proneural potential in a graded manner ﬁts nicely with Wolpert’s gradient scheme [3954]. In cut LOF discs, the marginal bristles become disorganized [886] probably because the interfaces that align the SOPs are gone (cf. Fig. 3.9d). Given Wg’s downstream links, it is not surprising that wg GOF clones induce ectopic margins when they arise anteriorly in the D or V region [1040] (albeit without turning on cut [2839]; cf. slimb LOF clones [2060, 2856, 4279]). Although the wg-on stripe straddles the D/V line [163, 3689], the D or V half of the stripe can be suppressed without appreciably affecting wing shape [1040, 2850]. A feedback loop apparently enables the remaining half-stripe to double its output of Wg [2839, 3690] (cf. Dpp [572, 1674, 4136]). Unlike the leg disc, where the wg-on state is inherited from the embryo [837, 4682], the nascent wing disc does not express wg. Only later does it activate and modulate wg expression in response to various spatial and temporal cues [834, 880, 4683]. At the margin, the chief cue for wg is the ap-on/off interface [3689]. This stimulus is mediated by the Notch pathway. Notch incites and sustains the marginal expression not only of wg [988, 994, 1074, 2839, 3689], but also of cut [988, 990, 994, 2839, 3089], vg [1250, 2216, 2254, 3089], and bHLH genes of the E(spl)-C [988] (cf. overall circuit [2252, 3089]). (The vg gene is regulated independently by Wg [2092, 3091, 4849] through a separate cis-enhancer [2219, 2254, 4684].) All pouch cells are competent to react in these ways [990]. For example, N GOF misexpression turns on wg and vg anywhere in the pouch [1040, 2219] but not outside it. The Notch links also explain why quashing Notch snuffs out Wg and Cut at the D/V line [459, 2548, 2839, 3689] and stunts pouch growth [1040, 3025]. Similar LOF effects are seen in Su(H) null discs [2253, 2453, 3089, 3826] and Su(H) null clones [994, 1040, 2079, 3089], implying that Su(H) relays the signal (N Su(H) target genes). Targets include bHLH genes at the E(spl)-C [991, 994], which may play an antineural role because deletions cause extra bristles [1794]. However, they seem irrelevant for the margin itself because deleting them causes negligible notching [991, 994, 1794], and m8GOF does not affect vg [2254]. (N.B.: Notching is the trait for which Notch, Serrate, cut, Chip, and scalloped were named, while wingless, apterous, and

IMAGINAL DISCS

vestigial denote wing reduction [470, 2561, 2940]). Clones that lack all 7 bHLH genes repress the cut stripe slightly when they overlap it, but groucho null clones turn cut off completely [2548]. Evidently, Notch activates cut not only via the E(spl)-C bHLH proteins (∗ ), but also redundantly via an unknown Gro-binding protein (“X”): “N {[E(spl)C∗ or X] and Gro} Y cut,” where “Y” is an unknown intermediate repressor [2548]. Based on their role in lateral inhibition (cf. Ch. 3), E(spl)-C bHLH proteins would be expected to directly repress ac and sc (vs. acting through cut). However, inhibiting cut seems sufﬁcient to totally derepress ac and sc [988], so such a parallel link is unlikely.

Fringe prevents Notch from responding to Serrate Notch is expressed fairly uniformly in immature wing discs [988, 1203, 1851, 2070, 2209, 2296], but its ligands Ser and Dl are not. 1. Ser is activated (and kept on) by ap in the D compartment in 2nd–3rd instar [885, 988, 1040] via a speciﬁc cis-enhancer [164]. Its downstream status (Ap Ser) is conﬁrmed by the ability of Ser GOF to rescue wing development in apLOF discs [2251, 2253]. In mid–late3rd instar, Ser fades from much of the D area and arises elsewhere (intensifying at the D/V border) [885, 2216, 4301] via different cis-enhancers that respond to other cues [163, 1250]. The only Ser null clones that affect the margin are those that reach it from the D side [885, 994, 1040], so Ser is only needed at the D edge of the D/V line. Misexpression of Ser in the V region induces “margin syndrome” (margin bristles, overgrowth, and activation of wg, cut, vg, and E(spl)-C genes) [885, 1040, 2092, 2216, 4029], implying that (1) all Vtype cells can respond to Ser and (2) the only cells that normally do so abut the V edge of the D/V line because Ser is tied to D-cell surfaces [988, 1074] and is silenced by a co-expressed inhibitor. The inhibitor turns out to be Fringe (see below) [1250, 3246]. Ser can also cause the syndrome when expressed near the D/V line on either side [988], implying that Fringe’s effects can be overridden near the border. Quasinormal triple rows (albeit V/V) only arise when Wg [2092] or Vg [2252] is also present. 2. Dl is transcribed throughout the disc as early as mid2nd instar – more strongly ventrally, and most intensely at the future wing margin [1074, 2253, 3246]. In mature discs Dl is upregulated in two bands that ﬂank the wg-on D/V stripe [988, 994, 1074, 2296, 2839] (cf. N

CHAPTER SIX. THE WING DISC

[1951, 2070]).

Thus, Dl’s expression pattern does not reveal as obvious a D-V signaling bias as Ser’s [357]. Nevertheless, Dl must be mediating polarized (V-to-D) signaling like Ser (D-to-V) because (1) Dl LOF clones at the D/V border nick the margin only when they reach it from the V side [994, 1074], (2) Dl GOF clones cause margin syndrome only in the D (and border) region [988], and (3) misexpression of Dl along the A-P axis (via ptc-Gal4) elicits ectopic margins, overgrowth, and cut and vg expression mainly on the D (vs. V) side [1250, 3246], although other aspects of the syndrome are induced on both sides [1074, 2092, 2253] and the effects with UAS-Dl lines are weaker than with UASSer [1074, 2092]. Apparently, Dl is modulated somehow so that transduction of its signal is blocked ventrally. The fringe (fng) gene was discovered ca 1991 [1990] via an enhancer-trapped lacZ at its locus that had an intriguing expression pattern in the wing disc [1992]. The inserted lacZ and the resident fng are expressed congruently with ap in the D compartment until late-3rd instar. Indeed, ap regulates fng [1992]. Like ap null clones, fng null clones in the D region that are near the endemic margin cause an ectopic margin along their edge (Fig. 6.7). However, the fng-on/off margins contain only D-type bristles in D/D symmetry. Evidently, fng acts downstream of ap to control trans-border signaling [1992, 4762], whereas D-vs.-V identities are regulated separately by a parallel (Dlw) branch in the circuit (Fig. 6.8). Dramatic evidence for this role comes from fng’s ability to rescue ap LOF winglets when it is artiﬁcially expressed dorsally [2850, 3158], resulting in full-size (albeit V/V symmetric) wings. Fng’s a.a. sequence suggested that it might be secreted [1992], and it is secreted in cell culture [2084] and to some extent in vivo [3246]. However, it only affects Notch in the Golgi [3000], and its effects in vivo are cell autonomous [2253, 3246], so its role here is intracellular. Indeed, Fng may be tethered to Notch during maturation [2096]. Fng turned out to be a toggle that switches the Notch receptor from one responsive state to another: Serrate cannot activate Notch in the presence of Fng [1250, 2253, 3246]. How does Fng interfere with the Ser-N interaction? Fng glycosylates Notch in the Golgi [510, 1268, 2904, 3000] before Notch reaches the surface (cf. earlier hints to this effect [1551, 2070, 2253, 4835]), and the added sugars increase Notch’s afﬁnity for Delta [510, 3246]. The same modiﬁcation

165

may make Notch refractory to Ser [360, 3245]. The Fng-N interaction seems to be direct [357, 510, 1250]. Fng interacts with EGF repeats 24–29 in Notch’s extracellular domain (cf. Fig. 2.3) [990]. There is no known counterpart of Fng in the V compartment that damps Dl-N signaling (while facilitating Ser-N signaling?) [1250], and it remains unclear whether such a doppelg¨anger must exist [360, 1944]. Scabrous can act in this way when artiﬁcially expressed [2462], but its normal expression at the margin occurs too late for it to be playing this role. The only sure conclusion is that the Deaf-Speakers/Mute-Listeners Trick is as instrumental for the D-V axis as it is for the A-P axis – albeit in a different way: 1. The D-V axis has two short-range signals (Ser and Dl) and hence two kinds of selective deafness, whereas the A-P axis has only one (Hh). In this regard, the D-V strategy is a vivid incarnation of Meinhardt’s Boundary Model [2808]. 2. The D-V axis enforces deafness at the cell surface (D cells cannot hear Ser because Fng modiﬁes Notch), whereas the A-P axis does so at the genetic level (P cells cannot hear Hh because En prevents expression of the transducer Ci). Although these strategies are equivalent developmentally, they must have had different origins evolutionarily. Regional suppression of a transducer like Ci should be trivial to evolve, but where on earth did a “deafness-promoting factor” like Fng come from? Like Numb, another regulator of Notch (cf. Ch. 2), Fng appears to have acquired an abstract switching function that is applicable in any context [1988]. Indeed, Fng also constrains Notch signaling to a compartment boundary in the eye (cf. Ch. 7), where it has other upstream regulators instead of ap. Hence, Fng’s fame as a versatile “boundary gene” [1988, 1992] is well deserved. Strangely, for Fng to block incoming Ser signals, the receiving cell itself must express Ser or Dl: when a fng-on cell’s Ser and Dl are both removed (dorsal Ser null Dl null clones), it can respond by turning on cut [2839]. This mystery exposes our ignorance about how Notch interacts with its ligands (sterically and stoichiometrically) under normal conditions [1944].

The core D-V circuit plugs into a complex network Figure 6.8 summarizes our basic understanding of D-V cross-talk and fate assignment. It incorporates the

166

IMAGINAL DISCS

following links, all of which are well documented, except for 1 and 5: Link 1:

Link 2: Link 3: Link 4: Link 5:

Link 6:

{Ap and Chip} Dlw? Dlw’s spatial expression is unknown, but its LOF and GOF effects imply allegiance to the D (ap-on) compartment [4325]. {Ap and Chip} Ser. Many other factors also regulate Ser in the wing [163, 1250]. {Ap and Chip} fng. No other regulators of fng are known in the wing [1988, 2851]. Fng {Ser N}. This link deafens D cells to their own signal [1250, 3246]. Fng {Dl N}? Fng dramatically increases Dl-N binding in vitro [510], but the extent to which it potentiates Dl-N signaling in vivo is less certain [2253]. Normally, Dl can only turn Ser on in the D area, but when Fng is put into the V area (by a fng GOF mutation or by ptc-Gal4:UAS-fng), Dl can activate Ser there as well (via ptc-Gal4:UASDl) [3246], a result that clearly implies Link 5. However, this effect (especially as manifest in ectopic bristles) typically extends only a short distance into the V half of the pouch (wg activation can extend farther [510]) and hence may stem from border peculiarities (Links 7–11 and 16–18). If Link 5 were a more general feature, then excess Fng ventrally should enhance the response to Dl, but such wings actually grow less (e.g., fng D4 GOF heterozygotes [1992]), not more [2253]. Even more telling, perhaps, is the fact that excess Fng fails to exacerbate Dl-mediated signaling during CNS neurogenesis [1250]. {Ser or Dl} N wg. Notch signaling only activates wg within the conﬁnes of the wing pouch [994, 1074, 2253]. This restriction apparently stems from the same proximal vs. distal dichotomy that operates in the leg [157] (cf. Ch. 5), with Teashirt and Hth-Exdnuc dimers governing the proximal domain [793, 2937, 3104] and suppressing key genes needed for distalization [678]. However, the boundary may instead be enforced by the Iroquois Complex [1060].

Omitted from the sketch in Fig. 6.8 are other components (e.g., strawberry notch [895, 2662]). Other connecting links (below) have been seen under various LOF and

GOF circumstances. Why certain links only operate at speciﬁc stages remains a mystery [981]. Link 7:

Link 8: Link 9:

Link 10:

Link 11:

Link 12:

Link 13:

Link 14:

N {Ser and Dl}. Notch stimulates transcription of its ligands [988, 3246] (but see [1951]). Ser Dl. Ser activates Dl transcription [2253, 3246]. Dl Ser. Dl has a reciprocal effect on Ser [2253, 3246]. This reinforcement (Links 8 and 9 acting via Link 7) upregulates both ligands near the D/V border in 3rd instar [1988, 3246]. The two sides are interdependent because unilateral inactivation of N abolishes N activity on the other side [2838, 3689] (but see [2218] for D-V asymmetries and [2838, 3524] for exceptions). Dl Dl. Given that Dl can activate its own transcription in adjacent cells [2253], this link should cause Dl expression to spread like wildﬁre throughout the disc (cf. Minotaur Scenario; Ch. 5). Why it does not is unclear [2253]. {Ser or Dl} N. Excess ligand autonomously represses N (and hence wg and cut) in the signaling cells themselves [988, 1040, 2248, 2253, 2839, 4301] by an unknown (cell surface?) mechanism [357]. Dl is weaker in this regard [1074, 2092]. This link creates a single-cell version of the Deaf-Speakers/ Mute-Listeners Trick that allows oneway signaling [2251, 2850, 3158]. Its normal role is to block N in cells ﬂanking the cut-on (wg-on) stripe late in development [2839]. Cut {Ser and Dl}. This link enables the cut-on (high-N) stripe to create a gap in the wider ac-on (high-Ser-high-Dl) band and thereby split it into two parts (Fig. 6.8c) [988]. Cut wg. Expression of wg is maintained (but not initiated) by Cut [2839] (but see [882]). Not surprisingly, the wg-on and cut-on bands coincide [882]. The wg-on band looks wider with a lacZ insert [353], but this illusion is attributable to perdurance of the β-gal product. Wg wg. A paracrine feedback loop maintains the width of the wg-on stripe [2839, 3690] (but see [2839]).

CHAPTER SIX. THE WING DISC

Link 15:

Link 16:

Link 17:

Link 18:

Link 19:

Link 20:

Cut? {Wg target genes}. Wg-secreting cells cannot respond to their own Wg [2839], possibly because they also express Cut. Wg {Ser and Dl}. Wg has an autocrineparacrine ability to activate Ser and Dl [988, 2839]. Dsh {N target genes}. Although Dsh belongs to the Wingless transduction pathway (cf. Fig. 5.6), it can interfere with Notch signaling, probably by directly binding Notch [151]. N {Wg target genes}. The ability of Dsh to “short circuit” the Wingless and Notch pathways implies a reciprocal antagonism [356], which may involve sequestration via N-Dsh heterodimers. Nubbin {N target genes}. Nubbin is a POU protein that damps N target genes in the pouch [3092, 3103]. It may serve the sort of generalized “threshold-setting” function ascribed to Dad and Nkd for the Dpp and Wg pathways, respectively (cf. App. 6). Fng {Ser and Dl}. When Fng is misexpressed ventrally, it activates Ser and Dl at its on/off boundary, but there is no reciprocal activation of fng when Ser or Dl are misexpressed [2216, 2253, 3246]. This effect could be mediated by the loop of Links 8 and 9 [1250], but it likely just relies on loop 7 because Fng can activate the Notch pathway under steric circumstances that might be mimicked by supersaturation [2096].

Elements upstream of Wg are all expressed by mid2nd instar [164, 1074, 4543, 4683], but wg does not turn on at the margin until late-2nd [3104] or early-mid 3rd [2252, 3374, 3690, 4683] instar, and cut comes on even later (mid-late 3rd instar) [2839]. The reason for these delays is not known. A few other nuances should be mentioned (see [2251] for further riddles). Target genes (e.g., wg vs. cut) may need different thresholds of N activation [2839]. Responses may depend more on the relative amounts of ligand vs. receptor in conversing cells (i.e., a sharp vs. fuzzy boundary) than absolute amounts in either partner [1074, 2092, 2216, 2839]. Removal of ap function alone is not enough to fully convert a cell from D- to V-type identity (as assayed by vg induction) [4684], but it is unclear what other redundant factors help specify D-type

167

identity together with ap [2251]. Nor is it known whether V-type identity is a default state or is actively implemented by ap and Dlw counterparts [885]. If Dl and Ser are membrane bound (as generally assumed [1251]), then the dialog across the D/V line should create a wg-on stripe that is only two cells wide [1988]. In fact, however, the stripe is 3–6 cells wide [353, 2079, 3689, 3690] (cf. vg [885]) and can broaden to ∼20 cells when Nubbin is disabled [3092]. This greater-than-expected width could be due to (1) diffusion or transcytosis of N ligands [885, 3479], (2) perdurance of the wg-on state [3363], (3) signal relay [3923], or (4) a larger-than-realized zone of productive ligand-receptor overlap [3092]. None of these possibilities can be excluded at present. The same quandary applies to wing veins [989, 1951] and to the activation of E(spl)mβ in intervein cells ≥5 diameters away from the source of Dl [871, 984]. In summary, the basic chain of events along the D-V axis is: Ap (D region) {Fng and Ser} N (on V side of D/V line) Wg (diffuses in both directions) target genes, including Ac ( bristles) and Vg ( pouch), with N being activated differently on the D side (? Dl N). Metaphorically speaking, a “tent” (symmetric Wg gradients) is erected on a “pole” (activated N) that is planted at the edge of a territory (ap-on domain), much like the process along the A-P axis. Viewing the process in this way elucidates the old “Field Effect Mystery” of molecular genetics: how can genes affect regions where they are not expressed? In this case, the ap null phenotype involves loss of the whole wing – not just the D part where ap is expressed – because ap is needed to spark a chain reaction that builds the entire wing [4683]. The symmetric Wg gradients lead to nearly identical patterns in the D and V halves of the pouch, which makes sense because the two surfaces must match perfectly to make a ﬂat airfoil. The slight differences that do exist (e.g., bristle types; cf. Fig. 6.7) are made possible by a gene (Dlw) that toggles the mode (D- or V-type) that cells use to interpret their gradient.

The wing-notum duality is established by Wg and Vein What causes Ap to be expressed in the D region in the ﬁrst place? Here, we encounter a queer irony. Whereas Wg is under Ap control during late stages of wing development, the opposite is true when the wing primordium ﬁrst forms. Unlike the leg disc where wg stays on in one subregion throughout development, wg expression in the wing disc changes with time (Fig. 6.2) [880]. Wg is ﬁrst

168

IMAGINAL DISCS

a Wing disc

b Eye disc

wg- ON

i

A

vn- ON ( vn (

wg or

LOF

iv N

V

LOF

Egfr

ii W

vn

E

A

GOF

LOF

wg(total)

wg

or

N

iii

vn

?

LOF

GOF

or Egfr

iii

GOF

W N

P

A

P

GOF

ii

V

D

i Egfr- ON? ( N ( N- ON

D

W

A

A

N Deficiency & duplication Deficiency only

c Homeosis

d Transdetermination

Wing

Genital

Proboscis

LOF

wg

GOF

wg

or GOF

vn

(total) Leg

Antenna or Palpus

Proboscis

Wing

Notum GOF

wg

or LOF

vn

(lost) Notum

Eye

Haltere

CHAPTER SIX. THE WING DISC

detectable in early-mid 2nd instar [880, 2252, 3104], when the wing disc contains ∼100 cells [4683]. At this stage, wg is transcribed in ∼10 cells that occupy an anterior-ventral sector (as in the leg disc) [207, 880, 4543, 4683]. Shortly thereafter (mid-late 2nd instar; ∼200 cells) the wg-on domain expands to span the ventral third of the disc [2252, 2253, 3089, 3104], while ap turns on in the remaining D portion [1074, 2251, 3104, 4683]. This complementarity is not absolute (overlap exists), but the following facts argue that it is probably causal (i.e., Wg ap).

169

When wg is suppressed during 2nd instar (by heating t.s. wg LOF larvae), the wing is often replaced by a mirror-image copy of the heminotum (Fig. 6.9) [880]. This duplication arises by in situ transformation [158, 159, 2929, 4543], rather than by tissue loss and compensatory growth [2015, 4683]. When wg LOF discs are examined around this time, they are found to express Ap throughout the disc [4683], implying that ap is normally repressed ventrally by Wg (Wg ap) but can assert itself when Wg is removed.

FIGURE 6.9. Bipolar duality of the wing disc (i.e., wing vs. notum, a) and eye disc (i.e., eye vs. antenna, b), homeotic tendencies

in the wing disc (c), and how these tendencies may explain the wing-to-notum trend in transdetermination (d). N.B.: The key for a (ii–iv) and b (ii and iii) straddles the a/b dividing line, and prospective adult axes are given in the compasses above (right wing disc, left eye disc). The letter “N” in plain type here represents the heminotum, whereas an italicized “N” signiﬁes the gene Notch. a. Genetic control of wing-vs.-notum identity in the wing disc. i. Wing disc (gray) showing regions (solid black) where vein (vn, dorsal spot) and wingless (wg, ventral sector) are transcribed in 2nd instar. Both vn and wg encode diffusible signaling molecules. Although Vn and Wg could probably diffuse into one another’s territory and exert mutual inhibition, the chief link that appears critical is “Wg vn”. ii–iv. Abnormal discs resulting from LOF or GOF changes in vn or wg expression. ii. “N/N” double-heminotum phenotype resulting from wg LOF mutations. The wing portion of the disc has transformed to notal identity. Evidently, vn is expressed by default when Wg is absent. N/N duplications can also be evoked by skinhead LOF LOF [3371], vg LOF [2252], and N [886] (cf. Table 8.1). Although vnGOF has not yet been tested, it is assumed to also behave this way because (1) Vn is the only essential ligand for Egfr in wing disc development [3025, 3928, 4604], (2) N/N defects can be created by misexpressing Egfr (via omb-Gal4) [4543], and (3) wing-to-notum transformations are seen in Egfr GOF clones [4543]. iii. “W/W” double-wing phenotype resulting from misexpressing wg throughout the disc (via T80-Gal4:UAS-wg) [3025]. The notal portion of the disc has transformed to wing identity. Apparently, Wg suppresses vn within the dorsal domain (Wg vn) and hence triggers the “wing subroutine” there as well as in wg’s native (ventral) domain. A partial W/W trait is rarely seen with vn LOF alleles (omitted), which instead typically cause notal loss (as in iv) [3801, 3929]. Its rarity is likely due to a tendency of wg to remain off in the absence of Vn. iv. “W/-” (notumless) phenotype resulting from vn LOF mutations [4543] or wg misexpression throughout the pouch (via sd-Gal4:UAS-wg) [2252]. The difference in wg GOF effects (iii vs. iv) may be due to a threshold of Wg concentration below which vn can be turned off without inducing wing development. Forcing wg to be expressed along the A/P border (via dpp-Gal4:UAS-wg) can yield either outcome (iii or iv) [2252, 3025, 3104]. b. Genetic control of eye-vs.-antenna identity in the eye disc (cf. Fig. 7.1). i. Eye disc (gray) showing the region (dark gradient) where Notch is transcribed in 2nd instar [2353]. Egfr’s transcriptional area is unknown; it may complement Notch’s. ii. “A/-” (eyeless) phenotype caused by LOF conditions of the EGFR pathway [2353] due to overexpressing the EGFR inhibitors anterior open, tramtrack, or Bar-C (also caused by wg GOF ). See Ch. 7 for “early eye” genes whose LOF alleles cause a similar phenotype. GOF conditions in the Notch pathway (N GOF ) can cause smaller, abnormal eyes (but see [2362]). iii. “A/A” doubleantenna phenotype resulting from LOF effects (“DN” = dominant negative) in the Notch pathway (N DN , Dl DN , Ser DN , or mastermind DN , but not Su(H)DN or m8DN ) or from GOF effects in the EGFR pathway (Egfr GOF , spitz GOF , Ras1 GOF , dRaf GOF , or pointed GOF , but not dMAPK GOF or dMEK GOF ) [2353]. The eye portion of the disc has transformed to antennal identity. A comparably complete transformation of the opposite kind (“E/E”) has never been found (but cf. Ch. 8). c. Summary of data from a.ii–iv in terms of histotype identities. Cells can undergo a wing-to-notum switch in two ways (thicker arrow denotes greater likelihood) but a notum-to-wing switch in only one way. d. Switches in histotype fate seen during long-term culture of disc fragments. Double-tipped arrows mean that switching can occur in either direction (size of arrowhead denotes relative frequency). Top-to-bottom order reﬂects the favored ﬂow of events, so the notal fate is a “sink” for the system. The tendency of wing tissue to transdetermine to notum (d) may be due to the ease with which such a transformation can occur homeotically (c). “Proboscis” appears twice (as per original data [1421]), which poses a paradox for any scheme that tries to rank histotypes hierarchically. Although no arrow is shown from eye to antenna, such switches probably do occur during culture because (1) antennal fragments duplicate themselves while eye fragments regenerate antennae [1404, 1406, 1423, 3810] and (2) LOF mutations in various genes transform eye to antennal tissue (but not vice versa; cf. Table 8.1) [2362]. Third- instar vn LOF leg discs can transdetermine to wing (not shown), even though mature vn LOF larvae lack wing discs, because they bypass the 2nd-instar need for vn [4768]. Schematic in a.i is based on [3928, 3929, 4543] for vn-on and [2252, 2253, 3089, 3104, 4683] for wg-on. Illustrations in a.ii–iv and b.iii are sketches of discs pictured in [2252, 3104, 4543] (a.ii), [3025, 3104] (a.iii), [2252, 3929] (a.iv), and [2353] (b.iii). Panel b.i is based on [3382], and d is redrawn from [1421] with one arrow omitted (from unidentiﬁed tissues to a genital state). See also App. 7.

170

The heminotum is the most dorsal derivative of the wing disc (cf. Fig. 6.1), so the wg LOF phenotype is analogous to D/D wg LOF legs (cf. Fig. 5.5) [4683]. Conceivably, Wg might be playing a ventralizing role as in the leg disc (Wg dpp), and wg does turn on in a leg-like ventral sector [880, 2251, 4683]. The parallels between wg’s action in wing vs. leg discs extend no farther, however. The leg disc has no bona ﬁde D or V compartments (cf. Ch. 5) [4076], and the wing disc only uses the “Wg ap” link brieﬂy [4684]. The wing-notum dichotomy that Wg sets up may not stabilize until later when other circuits involving vg (see below) assume responsibility for its maintenance. To avoid confusion with D/D effects inside the wing per se (cf. Fig. 6.7), this phenotype will be termed “N/N” (i.e., double notum). (“N” can also mean Notch protein, but not when used here with a slash mark. “W” will be used to designate the wing.) An N/N phenotype is also seen in vg LOF strains [2252] and can be created by heat-pulsing t.s. N LOF larvae during the same sensitive period as t.s. wg LOF (2nd instar) [886]. Evidently, wg, N, and vg collaborate to initiate the wing anlage at this time [885, 2219, 2252−2254]. If they fail to do so, then the prospective wing tissue reverts to a notal fate. The nature of this 3-way partnership is obscure but probably involves some of the links that later operate at the D/V border [4463]. Aside from wg, N, and vg, another element shared in common by the “wing initiation” and “D/V border” subcircuits is Egfr [207, 3025]. Egfr encodes the Drosophila Epidermal growth factor receptor (a.k.a. DER, Torpedo, Ellipse, or Flb) [812, 3484, 3535, 3536]. Egfr is a canonical receptor tyrosine kinase (RTK) [2573, 3464, 3779, 3780]. The EGFR pathway (discussed in a later section) must dictate notal identity because ligand-independent EgfrGOF clones make notal tissue and induce ap expression even when located in the wing [4543]. Egfr has four known ligands [1291, 3830, 4556, 4604] – all of which have an EGF motif: Gurken (a TGF-α relative that only acts in oogenesis) [3086, 3120], Spitz (also a TGF-α cousin) [3711], Argos (a competitive inhibitor) [1294, 1911, 2067, 3828], and Vein (an agonist that has an Ig-like motif) [3801, 3929, 4808]. All four ligands function in the D-V axis of the embryo [1529, 3085, 3758, 4117, 4557], where Spitz sets the ventral limit of the leg disc [1572, 3536]. A ﬁfth possible ligand (similar to Spitz) named “Gritz” has recently been identiﬁed [204, 2108, 4558]. Is any of the embryo’s D-V circuitry incorporated into the D-V axis of the wing disc? Apparently not. The expression of Vein (Vn), the wing disc’s chief Egfr ligand

IMAGINAL DISCS

[3025, 3928],

is not inherited from the embryo like en (cf. Fig. 4.4) [3929]. Rather, Vn is deployed in the notal region during 2nd instar [3928, 3929, 4543] – roughly the same stage when Wg arises in the wing area (Fig. 6.9) [880, 2252, 3104]. The “Egfr wg” link (deduced from effects of EgfrGOF LOF and dRaf clones [207]) can thus be rewritten as “Vn wg”. This link could allow Vn to deﬁne the wg-on area, but Vn evidently does not do so because wg is expressed normally in vn LOF discs [4543]. These same discs do not express ap, so Vn is required for ap activation (Vn ap) [4543]. Because Vn is secreted [3801], the Vn-Wg relationship could theoretically work like the Dpp-Wg “seesaw” in the leg disc (cf. Ch. 5). Indeed, Vn does “rebound” in the absence of Wg: 2nd-instar wg LOF discs have an extra spot of Vn where the Wg spot would have been [4543], and they go on to show N/N symmetry in other features as well. Added to the previous “Vn wg” link, this “Wg vn” link completes the seesaw, or so it seems. Strangely, however, Wg does not rebound in the absence of Vn: vn LOF discs lack notal tissue, but no wing tissue develops in its place [3929]. This “notumless” (“W/-”) phenotype is also seen in (1) Egfr LOF mutants LOF [3025] (cf. the “V/-” subclass of dpp legs; Fig. 5.5), and GOF (2) wg discs where wg is expressed throughout the pouch (via sd-Gal4:UAS-wg) [2252]. The sensitive period for this phenotype is 1st and early-2nd instar (based on a t.s. vn LOF allele) [3929, 4543]. When the entire disc (vs. only the pouch) is ﬂooded with Wg (via T80-Gal4:UAS-wg), a “W/W” phenotype (two pouches but no heminotum) arises (Fig. 6.9) [3025]. In this case, Wg cannot be totally suppressing Ap (Wg ap) because enough Ap must persist to activate the D/V border circuit for pouch formation. A partial W/W phenotype is rarely observed in vn LOF mutants [3801, 3929]. Its sensitive period is 2nd and early-3rd instar [3929]. The key facts presented thus far are summarized below: 1. Each ligand normally governs a separate region. Vn rules the notum, while Wg rules the wing. 2. The GOF effects are reciprocal. Forcing the Wg pathway into an on state can shut Vn off within its own domain (Wg vn), and vice versa (Vn wg). 3. The LOF effects, however, are not reciprocal. Removing Wg from its normal domain results in Vn being expressed there, but removing Vn from its normal domain does not result in Wg being expressed there.

CHAPTER SIX. THE WING DISC

Why does Vn, but not Wg, rebound when its opponent is removed? Presumably, vn has a ubiquitous cis-enhancer (or basal promoter) stronger than that of wg. (In the leg disc, both dpp and wg must have strong controllers.) The “default state” of the wing disc would thus be vn-on, with the wg-on area being superimposed on this baseline by (1) an upstream regulator turning wg on ventrally and (2) Wg turning vn off when it reaches a certain level. An obvious candidate for wg’s initial regulator is Hh, although it is unclear why wg is not also activated dorsally and continually until pupariation. Interestingly, only the dorsal (wing, haltere, eye) discs require vn [80, 3291, 3928, 3931, 4768], while wg is needed in all the major discs [880, 1163, 1780, 1781, 3732].

But Vestigial and Scalloped dictate ‘‘wingness’’ per se Vestigial is expressed in an even more restricted subset of discs than Vein – viz., the wing and haltere discs [4681]. Indeed, it is emblematic of their inception in the embryonic ectoderm (cf. Ch. 4) [827, 1571, 1572, 4681]. Both discs transcribe vg uniformly until 2nd instar, when transcription subsides outside the pouch [4683]. The launching of pouch development is tantamount to creating a proximal-distal axis for appendage outgrowth [4682, 4684], and vg has been suspected of encoding “wingness” because (1) it turns on throughout the pouch [4683], and (2) it is required autonomously by wing cells for growth [3960]. Expression of vg in the pouch is regulated by two main cis-enhancers [982, 2254]: 1. The “boundary” enhancer (vgBE) is located in vg’s 2nd intron [2219]. It binds Su(H) and keeps vg on at the D/V border in response to Notch [2218, 2219]. Initially, it also needs Wg [2254]. 2. The “quadrant” enhancer (vgQE) is in vg’s 4th intron [2219]. It binds Mad [2217], Ventral veinless (Vvl) [703], and probably Arm-Pan [2254]. It turns vg on in the rest of the pouch in response to Dpp, Wg, and Vvl [703, 2217, 3091, 4849], with input from Vg itself as well [2254, 3933]. Expression of vgQE vanishes in clones that are singly mutant for Mad LOF [2217], arm LOF [3091, 4849], or vvl LOF [703], so vgQE must use a combinatorial rule like the “{Dpp and Wg} al” rule that governs al in the leg disc (viz., “{Dpp and Wg and Vvl} vg”). The vgQE is off at the margin because Vvl is absent there – a lack that is due to Wg (Wg vvl) [703] and, ultimately, Notch (N wg) [994, 1074, 2253]. Interestingly, vgQE is ﬁrst activated at the

171

center of the wing disc (analogous to al) before its expression broadens to ﬁll the pouch [2219, 2254]. The suspicion that vg might be a “master gene” for wing development was conﬁrmed in 1996 by showing that it can coerce cells in foreign (leg or eye) discs to make wing tissue when it is ectopically misexpressed [2219]. This “magic trick” can also be performed by wg GOF [2092, 2252], but vgGOF is more adept at turning on downstream wing marker genes (e.g., nubbin) [1686, 2252]. Moreover, when wg GOF induces ectopic wing tissue, vg turns on at those sites [2092], whereas the reverse is not necessarily true [2219] (cf. Table 8.1). The vg gene is autoregulatory (vg vg) insofar as Vg activates both vgBE and vgQE [1686, 3291]. However, Vg is ineffective without its auxiliary protein Scalloped (Sd) [1686, 3291, 3936]. Vg and Sd are expressed congruently during wing disc development [4683], and vg and sd maintain one another’s transcription: “Vg sd” and “Sd vg” [3291, 3936, 4463, 4683]. The former link also operates outside the wing disc, although the latter apparently does not [2219]. Vg is a nuclear protein without any known DNAbinding motif [4681]. Sd is a member of the TEA family of transcription factors [623, 1029, 2010], and as such it binds tandem 9 b.p. sequences that conform to the consensus of its human ortholog [1686, 2940]. Vg and Sd bind one another [3291] but do not form homodimers [3936]. In the absence of Sd, Vg is found primarily in the cytoplasm, but Vg localizes to the nucleus when Sd is present, implying that Sd escorts Vg or retains it after nuclear entry (Fig. 6.10) [1686, 3936]. The relative amounts of Vg vs. Sd are critical [3291, 3936, 4463]. For example, excess Sd reduces target gene activity [1686], and imbalances in either direction cause similar phenotypes [3936] (cf. the Ap-Chip story above [1217]). When the Su(H)-binding site in the vgBE DNA is replaced with binding sites for Ci-155, the reporter gene switches from Notch to Hh control as expected [1686] and turns on in a stripe at the A/P (vs. D/V) border. Oddly, however, it is not expressed in the leg disc, despite the presence of a similar A/P border. Why not? Conceivably, Vg-Sd binding sites elsewhere in the enhancer bind a repressor when Vg-Sd dimers are absent (as in the leg disc), and this repressor blocks activation by Ci-155. The repressor could be Sd itself, or a co-repressor recruited by Sd (Fig. 6.10f). Whatever its nature, the inhibitory agent must be powerful enough to prevent trans-activation by Ci. Any cis-enhancer having Vg-Sd binding sites would thus operate according

172

IMAGINAL DISCS

a vg- ON =

b sd- ON =

A

A

H E N

Key

A eye disc H E N

H E W L

W L

N

and

W wing disc

c

L

Vg

leg disc

d

Sd

e

f Vg

Sd

Vg

move

? OFF

Vg

Sd

ON

Sd

OFF

bs

bs

bs

etc.

etc.

etc.

Non-wing

"Wing" state

Non-wing

FIGURE 6.10. The “Venn Overlap Rule,” as manifest in the control of wing blade identity by vestigial (vg) and scalloped (sd).

a, b. Regions where vg and sd are normally transcribed (black areas). These regions are plotted on a schematic map (key in a) where the derivatives of eye, wing, and leg discs are outlined by thick lines, and subregions (A = antenna, H = head capsule, E = eye, N = notum, W = wing) are partitioned by thin lines. a. The vg gene is transcribed throughout the wing and in small areas of the hinge and notum (not shown; cf. Fig. 6.2) [4681]. b. The sd gene is transcribed throughout the vg-on territory, as well as in eye and leg discs [623]. Although expression in eyes per se is well documented [623], the antennal, head, and leg areas plotted here are only guesses based on the ability of ectopic vg to induce wing tissue wherever sd is already on [2219, 3936]. c. Venn diagram of the logic. Only when Vg and Sd are co-expressed (black area of overlap) will wing tissue be formed. Put more generally, the Venn Overlap Rule states that when two or more genes interact cooperatively, they only activate target genes where their domains of expression overlap [2353]. d–f. Illustrations that show how the rule is implemented at a molecular level. d. When a cell expresses vg alone, Vg resides mainly in the cytoplasm [1686, 3936]. Even those Vg molecules that do enter the nucleus are impotent because they cannot bind DNA without help from Sd. e. When both Vg and Sd are present, Vg binds Sd [3291] and the Vg-Sd complex is found mainly in the nucleus. Nuclear localization could be due to escorted import (as shown) or to selective retention after import. The Vg-Sd complex binds a DNA sequence (gray box) near target genes like blistered (bs) [1686]. Some of those genes (not bs itself ) execute wing identity. Whether the Vg-Sd dimers activate transcription directly or merely allow other trans-activators to turn on bs, etc., is unknown. f. When a cell expresses sd alone, Sd may bind some of those same sites (but see [1686] sequel). In the absence of Vg, trans-activators like Ci cannot turn on such target genes [1686]. The reason for this “locked off” state may be that Sd recruits a strong co-repressor (“?”). See also Fig. 6.14f and App. 7.

CHAPTER SIX. THE WING DISC

to a Boolean rule: “Turn on ONLY if you are in the wing pouch AND you are prodded by the trans-activator of a signaling pathway; otherwise remain off, no matter what activators try to turn you on!” The “locked off” state is fundamentally different from the “passive off” state (Fig. 6.10), and it has different implications for evolution (cf. Ch. 8). This “locked off” trick is evident in the Pangolin story (cf. App. 6) [3147], although neither Pan nor its partners preside over a discrete body region as Vg does. Pan (≈ Sd) locks its target genes in an off state by recruiting Groucho when Arm (≈ Vg) is absent [692, 2496]).

Straightening of the D/V border requires Notch signaling and Ap An old issue in embryology is the “Euclidean Ruler Problem”: how do cells draw lines? Any system – be it sentient or cybernetic – that builds complex structures must be adept at drawing lines, squaring angles, and calibrating distances. Unlike humans, cells are handicapped as geometers because they cannot be surveyors. Except for neurons, cells have no “eyes” or their equivalent, so they must rely on local cues. Epithelial cells do not normally organize themselves into neat ﬁles [4641], but on occasion they must. Wing veins are a case in point. Somehow, wing cells have mastered the ability to form straight lines that become veins. Vein 1 forms along part of the D/V boundary, so this border may offer clues about vein formation in general. Two possible alignment strategies were discussed before for the A/P boundary (Fig. 6.5): the Selector Afﬁnity and Border Guard schemes. Which of them is used at the D/V boundary? In 1999, this question was analyzed by three different teams – Micchelli and Blair in Wisconsin [2838], Rauskolb et al. in New Jersey (Rutgers) [3524], and Mil´an and Cohen in Heidelberg [2850]. Overall, the results argue that both types of mechanism play a role. Notch signaling must be involved because 1. The D/V line becomes ragged when t.s. N LOF larvae are heat-pulsed or a dominant-negative N allele is expressed [2838]. 2. Some Nnull clones cross (D to V or V to D) when they arise at the border [2838, 3524], and the same is true for clones that express a “deaf” N construct (truncated intracellular domain) [3524]. In contrast, clones whose N is constitutively activated never cross [3524]. 3. Dl LOF Ser LOF clones can cross the border in either direction [3524].

173

4. Nnull clones only cross when N signaling ceases on both sides of the line [2838, 3524], and the same is true for Dl LOF Ser LOF clones [3524]. 5. Crossing also occurs with fng LOF (D to V) and fng GOF (V to D) clones that arise near the boundary [3524]. Because ap still functions in all these wings, ap cannot sufﬁce for alignment. Su(H) null clones respect the D/V line and the line stays straight even when it traverses wg null tissue, so neither Su(H) nor Wg is mediating N’s effects [2838]. The above results suggest two types of afﬁnity: (1) a “border” afﬁnity for N-stimulated cells and (2) a “default” afﬁnity for all other cells. N-activated cells evidently align to form a marginal band in the midst of an otherwise homogenous pool of blade cells [2838, 3524]. In 2001, this Border Guard Model was validated by O’Keefe and Thomas in San Diego [3158]. By expressing Fng and a D-speciﬁc integrin in the D part of ap LOF wing discs, they enabled these discs to make a straight margin of wg-on cells despite an apparent lack of any D-type cells. The rescued discs went on to make symmetric V/V wings that were otherwise remarkably normal in appearance. In wild-type wings, Ap may control a subtler afﬁnity within the border zone [2838]: ap-on and ap-off cells segregate into ∼2-cell-wide bands within the ∼4-cell-wide N-activated zone [3524]. Cells can evidently drift into or out of the border zone by jostling during growth, but D-type cells cannot become V-type or vice versa [2838]. Evidently, this is how D vs. V lineages maintain the apon/off compartment boundary [352].

Straightening of veins may rely on similar tricks Similar arguments were made earlier for the dpp-on zone at the A/P line. In each case, the border might be straightened by homophilic molecules that are downstream targets in the pathway. Interestingly, both pathways (Dpp and Notch) cooperate in vein development. This “double dose” of adhesivity could explain why extra vein tissue tends to (1) coalesce with existing veins to create branching networks, (2) form smooth curves [3372], (3) adopt narrow ∼4 cell widths [1368], and (4) acquire smooth edges [3624]. These traits are obvious with blistered LOF clones [1312, 2907, 3624] but are also evident in wings that are dppGOF [980], EgfrGOF [517, 2493, 4604], heartlessGOF [3558], net LOF [206, 320, 466, 4189, 4307] (cf. daughterless LOF [2741]), plexus LOF [206, 2742, 4307], rhoGOF [1643], rolledGOF [517,

174

spitzGOF [3800], sprouty LOF [681], veinGOF [4604], etc. [3076, 3558, 4794]. Whenever extra veins are elicited, they tend to arise midway between existing ones. These midway zones are distinctive in their timing of mitoses [3813], D-V annealing [431, 1312], and gene expression [4831] (cf. Fig. 3b of [4397]), so they may have special biases. The “Paravein Hypothesis” asserts that ectopic veins emerge there as an atavistic unveiling of ancient “paraveins” (old veins now lost) [320, 1368, 3237, 4307]. However, the channeling could be due instead to a mundane “Stick-and-Straighten Scenario.” To wit, if (1) excess vein tissue arises randomly and (2) contact with extant veins is likely within, say, one third of an intervein, then “rivulets” in the outer thirds will merge with extant veins [4189], while each leftover “puddle” in the middle will self-assemble into a new vein. This conﬂuence idea does not preclude some corridors being less hostile to vein formation for other reasons [984], nor does it rule out other dormant relics from ancient circuits. However, the latter must be hard to awaken because Drosophila’s vein pattern has been stable for ≥23 million years [1625]. The inferred ability of vein tissue to cohere and linearize has its own sort of evolutionary signiﬁcance. It is evidently a robust way of ensuring perfect veins from crude areas [3624]. Cell rearrangements have likewise been implicated in the straightening of bristle rows (cf. Ch. 3) [1800, 2449, 3603]. Intervein cells manifest a different kind of adhesion [430, 431, 3472, 4664]. They “zip” the D and V wing surfaces together by adhering with one another across the “transalar” lumen that is created during evagination [1312, 1313, 2907]. 2713, 3624],

Two cell types predominate in the wing blade: vein and intervein Wing veins serve as hydraulic pipes for wing expansion [1738, 4509], as spars for ﬂapping ﬂight [1116, 3015, 4749, 4750], and as conduits for nerves and tracheae [363, 1311, 3234, 4065]. The physical association of veins and tracheae [1368] is intriguing considering how many circuit elements are shared by vein patterning and tracheal branching [2576, 2679]: blistered [29, 268, 1646, 2907] (via separate cis-enhancers [3145]), colt [1738], Delta [766], dpp [4546], dumpy [4668], knirps [732], Notch [2574, 4082], rhomboid [4546], spalt [732, 2347], sprouty [681, 1665, 2321, 3558], ventral veinless (a.k.a. drifter [84, 85]) [337, 995, 2575], etc. Nevertheless, these parallels are perplexing because the styles of morphogenesis seem so different (e.g., polarization, movement, adhesion) [1312, 2679, 3729].

IMAGINAL DISCS

Excluding the margin and a few sensilla, there are only two salient cell types in the blade: vein (∼10% of the surface) and intervein (∼90%) [1312]. Vein cells differ from intervein cells in several key respects [1312, 2907]. 1. Density. Vein cells are more densely packed than intervein cells [1368, 2847], with appreciably smaller apical diameters [1312, 3015]. They secrete a thicker cuticle [4188], which gives the veins more tensile strength. D vs. V convexities in the veins give the wing a “corrugation” [2847] that resists curling. 2. Adhesion. Intervein cells differentiate basal extensions that help anneal the D and V surfaces [431, 1312, 1313, 4407]. In contrast, vein cells manifest little afﬁnity for the opposing surface, regardless of whether it contains vein (normal situation) or intervein (mosaic condition) cells [2907]. Instead, they form a lumen [1312, 1313, 3015]. This D-V cooperation during differentiation explains the transalar nonautonomy so often seen with vein-affecting clones [2847]. Nevertheless, veins can vanish [995, 1042, 1043] or form ectopically [4848] in one surface without affecting the other. 3. Death. Intervein cells die after the ﬂy ecloses from its pupal case, but vein cells survive [2077, 3015]. 4. Upstream controls. The vein state is the end result of a long cascade of signals (cf. Fig. 6.11). In theory, therefore, the intervein state could merely be a default [2742]. In fact, however, the selector gene that enforces it – blistered [320, 2907] – is actively turned on by Hh and Dpp in different interveins via separate cis-enhancers [3145]. Other regulators might include Daughterless (bHLH) [2741], Extramacrochaetae (HLH) [206, 913, 983], Net (bHLH) [206, 467], and Plexus (nuclear matrix protein) [206, 2742]. 5. Downstream effectors. The transalar adhesivity of intervein cells is attributable to higher levels of integrins [431, 504, 1313, 2907, 4664] and Dumpy (a huge adhesion protein) [4668]. In contrast, vein cells manifest higher levels of extracellular matrix components (laminin [1312, 1313, 3015] and collagen [3015]) and F-actin [1312, 1313], and have darker pigmentation [3624]. Vein cells express Delta but can differentiate without it [1951]. Because Blistered (Bs) has a MADS (DNA-binding) domain [29], it could establish the intervein state directly by activating “realizator” genes for cell shape, adhesivity, etc. [2907]. The vein state is controlled by the EGFR pathway (see below) and constrained by the distribution of rhomboid (rho) [320, 4556]. In the wing pouch, the expression patterns of bs and rho are complementary

CHAPTER SIX. THE WING DISC

(Fig. 6.2), except that rho-on stripes are narrower than bs-off gaps [320]. LOF-GOF analyses reveal a mutual antagonism (“rho bs” and “bs rho”) [320], although rho’s effects on bs are quantitative (except for L2 [320]), and initial expression of rho is normal in bs null discs [3624].

175

the receiving cell transports N-intra from its surface to its nucleus to turn on target genes (like two dogs sharing one bone; cf. Fig. 2.3) [3271]. Serrate may also serve as a ligand because removal of both Ser and Dl (in somatic clones) causes wider veins than the loss of either one alone [4859], but overexpressing Ser has no effect on the vein pattern [1951].

Veins come from proveins that look like proneural fields Veins widen to ∼10 cell diameters [984] when the Notch pathway is suppressed by Dl LOF [989, 1951, 2249, 3277, 4189] (cf. Dl DN [1951, 4207]), deltex LOF [1269], gro LOF [1794], H GOF [2657], kuz LOF [4025], mastermind LOF [437], N LOF [981, 984, 2462, 2847, 3025, 4189], shi LOF [3271], Su(H) LOF [989], or LOF alleles of bHLH genes in the E(spl)-C [989, 1794, 4256], although the E(spl)C is dispensable for L1 [991, 1794]. In the case of Dl LOF , the thickening is especially noticeable at the tips of the veins, which ﬂare into “deltas” [76, 1269, 4466] (whence its name [2560]). Conversely, veins are suppressed (i.e., shortened, interrupted, or eliminated) when the pathway is hyperactivated by DlGOF [1951], H LOF [117, 198, 989], NGOF [1951, 4161], or GOF alleles of bHLH genes in the E(spl)-C [2063, 2548, 4256]. These hyper- and hypoplasias imply that Notch’s role in vein formation is analogous to its role in SOP selection [989, 993, 4859]. That is, it might be whittling large areas to smaller size by preventing subsets of competent cells from adopting a desired fate. The competent cells in this case would occupy “proveins” (≈ proneural ﬁelds; cf. Ch. 3) [320, 1951, 3271] that are 4 to 5 times wider than the ﬁnal veins. When the Notch pathway is disabled, its “lateral inhibition” process apparently fails, and entire proveins form veins that are thicker than normal. The provein may extend ∼10 cell diameters on either side of each vein because Nnull clones make vein tissue when they fall within this range [993] (cf. Dl LOF , Su(H) LOF , and E(spl)CLOF clones [989, 1074]), and veins of this width develop when N and emc are simultaneously incapacitated [205]. Moreover, these are the sorts of widths seen for S-phase zones (labeled with BrdU) at 12 h AP [3813], so proveins may be real entities [981]. Dl and N both have haplo-insufﬁcient (Nnull /N + or null Dl /Dl + ) thick-vein phenotypes [3022], but the double heterozygote (Nnull /N + ; Dl null /Dl + ) looks wild-type [990], implying that Dl and N must balance for the pathway to function properly. This stoichiometry is starting to make sense now that the extracellular (N-extra) and intracellular (N-intra) parts of the Notch molecule can be tracked separately. When Dl binds N, the signaling cell ingests both its own Dl and its neighbor’s N-extra, while

But the resemblance is only superficial Despite the obvious parallels, the Dl-N circuitry cannot be operating here like it does in SOP selection. The reason is that veins are presaged by sharp stripes only ∼1–2 cells wide before the Notch pathway is deployed in a reﬁnement role [320]. For example, rho is expressed in a narrow stripe in the L2 vein area at pupariation [320, 2847, 4191], whereas Dl is not even detectable there until 22 h later [1951]. In fact, rho acts upstream of Dl (Dl is repressed in rho LOF vn LOF wings and turns on ectopically in rhoGOF wings [320, 989]). How can any gene turn on in a thin stripe before lateral inhibition has reduced proveins to a mature width? There is no obvious solution to this “Athena Enigma” (referring to the goddess born in adult form), but there are a few clues [328]. In mature discs, Dl is expressed in stripes that are 3–6 cells wide (ﬁnal veins are ∼2–3 cells wide [984, 4831]) [320, 1951, 2296], while N is expressed in a complementary pattern (i.e., in interveins and lateral proveins) [1203, 1951, 2209, 2296]. The critical period when Dl and N are needed for width control is 22–30 h after pupariation [1951] (but see earlier effects [4189]: their Fig. 5k). At that time, the Dl-on stripes are unchanged in width [1951], while the N-on zones have shrunken to ∼2–3 cell-wide ﬂanking stripes (with a residual basal level elsewhere). The Notch pathway is activated in the latter stripes [3271]. Effectors such as mβ must stop lateral provein cells from implementing the vein state [989], but how? Physical forces are probably involved [1951] because D and V surfaces are annealing at this time [1313], and surgical operations can induce vein fragments [2472] or reroute existing veins just before then (11–21 h AP). Even wild-type wings manifest a wide-vein phenotype when their D and V surfaces are prevented from annealing (by transplantation of disc fragments) [2847]. At present, the only ﬁrm conclusion is that Notch is regulating differentiation here, rather than determination [1951]. Thus, unlike its role in proneural ﬁelds where Notch shrinks stripes to lines and points (cf. Ch. 3), Notch is charged here with maintaining pre-existing lines. It must keep the widths narrow by enforcing

176

IMAGINAL DISCS

Gene expression patterns vein

Gene circuitry

3

distal

4 A

2

P

p

ro

xi m

gene has LOF effect

e

Hh

e

ara

kek1

vein

b

c

Hh ptc knot vein kek1

Ev Ev Ev Ev Ev

Dpp spalt kni ara caup

Ev Ev... Ev Ev... Ev...

rho

rho Ev... argos Ev Star Ev ? Egfr Ev Rl-act Ev bs Ev net Ev m Ev h Ev ac Ev

rho

net

vein

t ≈ 1 day

d

e

Rl-act

bs

rho

e

States vein intervein

Dl +30 N-act +30 m +30 rho Star Egfr Rl-act argos vein tkv dpp bs net vvl

+30 +30 +30 +30 +30 +30 +30 +30 +30 +30 +26

1

provein vein

5

A/P A

e

ON OFF

L5

activates inhibits e external signal ectopic vein o outside clone i inside clone

a

Key

al

Key

L2

B

L3

C

oo ooooooooooooooo i i i i i

i i i i i i

L4

D

L5

E

CHAPTER SIX. THE WING DISC

alignment in the face of mitoses, jostling, and other deviations within the plane and correcting any transalar mismatches that arise [320]: 1. Straightening. The Notch pathway is active in lateral provein, not vein, cells. Whatever homophilic adhesion molecules that it evokes would thus facilitate alignments on either side of the nascent vein. If vein cells themselves use a different adhesive protein, then each provein would be a 3-ply structure, where the ﬂanking bands might help “sandwich” and reinforce the central one for added rigidity. This hypothesis could be tested by monitoring cell movements. 2. Matching. An engineering problem that wings face (which legs do not) is the matching of two patterns that arise independently [3052]. A possible reason for the provein strategy, therefore, is that it allows “lastminute” adjustments of identities if veins on the D and V surfaces are out of register [4189]. Each intervein “zips up” from the middle outward [431, 1312], and mistakes may be “ironed out” by revising states of determination that are kept labile for just this purpose [1312]. This lability may explain how extra crossveins arise when the level of integrins (≈“transalar glue” [3158]) drops too low [936, 4664]. Removal of a vein’s D moiety typically blocks the V moiety from mak-

177

ing vein material [1359, 1643], although V/V wings have virtually normal venation [3158]. How transalar interactions cause fate switching at such a late stage is unclear. Again, cinematography might help, especially if D and V surfaces are contrived to have different vein patterns. Notch ﬁnishes its ﬁne-tuning chores long after the initiation phase has been completed. Vein initiation involves many different factors. As described below, each vein appears to have its own “board of directors” in the same sense that pair-rule stripes are managed by ensembles of different gap genes in the embryo [984, 4188] (cf. Ch. 4). In the discussion below, particular veins will be designated by number (1–5) or by “L1–L5” [4189], alluding to their longitudinal (vs. cross-vein) orientation. Intervein areas will be denoted by “iv” plus the numbers of the ﬂanking veins (e.g., “iv3–4”).

All veins use the EGFR pathway Another example of the Athena Enigma is found in the embryo [2617], where single ﬁles of mesectodermal cells turn on the bHLH-PAS gene single-minded (sim) on each side of the V midline [899, 4297, 4548] and then merge during gastrulation [3043, 3044, 4023]. Like rho-on preveins, these sim-on ﬁles (1) are perfected ab initio [1278], (2) also

FIGURE 6.11. Domains of gene or protein expression along the A-P axis of the wing pouch or pupal wing.

Data are organized in 5 panels based on stage (a–c: ∼0–6 h after pupariation = “Ev”; d and e: ∼30 h after pupariation = “+30”) or pathway (a Hh, b Dpp, c EGFR, d Notch). “Ev” stands for evagination, while “Ev . . . ” means ∼0–24 h after pupariation. Certain genes are presented in more than one panel. Except for Dpp and Hh (where protein levels are plotted) and Rl and N (where activated states are mapped), black areas denote transcription (cf. key, upper left). Degrees of expression are indicated by shades of gray or by graded slopes. Abbreviations: A/P (compartment boundary), A–E (intervein zones), L2–L5 (longitudinal veins 2–5). Full rectangles beneath L2–L5 (cf. key, upper right) are “proveins” [3271] (cf. preveins [377, 1312, 3015]), while squares denote mature veins (≈ 2–4 cells across [1312]), and ﬂanking areas are termed “lateral provein.” For genes whose LOF alleles cause ectopic veins, “i” or “o” denotes whether the vein is inside (autonomous) or outside (nonautonomous) the clone, and the span of symbols delimits the area where the effect is seen. Wiring at left ( activation; inhibition) illustrates some genetic interactions (see text or references below). See also App. 7. Genes or proteins (“∆” = veins missing in LOF ﬂies or clones; “≈” = mutations causing similar phenotypes; “DO” = details omitted): ac (achaete) [320], ara (araucan; ∆L3 [320]) [1536], argos [2715]∆ , bs (blistered) [984]∆ , caup (caupolican) [984]∆ (activated by Hh [2993], not shown), Dl (Delta) [205, 3271] (but see [989]), dpp (decapentaplegic) [980] , Egfr [1643] , h (hairy) [665], Hh (Hedgehog) [1208] , kek1 (kekkon1) [3020], kni (knirps; ∆L2; compare radius incompletus [320, 2617] and Fig. 4.2) [984] , knot [2894] , mβ (E(spl)mβ) [871, 984] , N-act (activated intracellular fragment of Notch) [3271] , net [466], ptc (patched) [2834] , rho (rhomboid; a.k.a. veinlet) [1643] , Rl-act (activated Rolled; a.k.a. MAP kinase [332]) [1643, 2715] , spalt (∆L2 [984] ) [2742] , Star [1643], tkv (thick veins) [2457] , vein (∆L4 [320] ≈ Egfr LOF [3624]; DO: gap at wing margin due to Wg) [4604] , vvl (ventral veinless) [995]. Interestingly, vvl is the only generic “vein gene” that is off in L1 (not shown). Possibly, vvl continues to be repressed by Wg at the margin [703] (cf. Fig. 6.2), just as ara and caup are repressed there by Wg (and N) [1537]. The format is of this ﬁgure is based on [320]. Nomenclature follows [1366, 4307] (but see [4065]). For a review, see [328].

178

IMAGINAL DISCS

EGFR pathway

Key binds activates inhibits switches state phosphorylates dephosphorylates

Spi ligand

es

s iffu

Vn

Argos "

socket

ligand Boss

P

proligand

P

Sev

Sev

Kek1

P

Star

receptor

cog?

Egfr

cle av ed

dimeric receptor

helper

Ligand Output

"

or

P

plug

Sevenless pathway

or

d

Egfr

P

P

cog

mSpi

dCbl

P

cog

helper

dShc

Rho

?

? enz & cog Csw

enz & cog

PLC

cog

cog

Drk

Spry

Sos enz

Gap1

? P

P

enz

cog Dos ? ?

enz

enz

Ras1 GTP

GGT1

Ras1 GDP P

enz

enz

Rap1

enz

PP2A

dRaf P

P

enz

enz

dMEK

PTP-ER ?

enz?

Ksr cog?

?

?

P

P

P

cyt. . nuc

P

dMAPK enz P

P

P

tr. factors

dJun activates

Pnt

Yan P

represses

Target genes

Instant Reset?

CHAPTER SIX. THE WING DISC

express rho [326, 330, 2405, 4192], and (3) widen or shrink like veins in response to LOF or GOF alterations in Notch signaling [2712, 2941]. Neither sim nor any other bHLH-PAS gene appears to be involved in wing development, but the coincident expression of rho suggests that the EGFR pathway, like the Dpp and Notch pathways, is a generic tool for line drawing. The EGFR pathway is outlined in Fig. 6.12 [3348, 3672]. Egfr is one of 8 characterized RTKs in Drosophila [1338, 2321, 2660, 3342, 3558]. The others are Breathless [1507, 2245, 2469, 3560], Derailed [612], dTrk [3475], Heartless [267, 1505, 3906], Insulin Receptor [1215, 1216, 4815], Sevenless [226, 428, 1677], and Torso [809, 4045, 4046]. All these RTKs have been shown, or are assumed, to “plug into” the same basic Ras-MAPK engine [1081, 1098, 1117, 1337, 1338, 3340]. Domain-swap experiments reveal differences in the potency of RTK kinase domains [1446, 3559], but no qualitative differences downstream. However, some elements are receptor-speciﬁc [1086] (e.g., dShc is used by Egfr but not by Sev [2623]). Thus, there is no obvious answer to the “Ras Speciﬁcity Riddle” [1291, 2647, 3590, 3790, 4245]: how can the same RasMAPK chain evoke different cell states at different times and places? The likely answer, in some cases at least, is that cells respond differently because they have been primed with different transcription factors [1688, 3942] so the RTK signal can only affect key promoters if other

179

factors are present [3874]. That is, cells use combinatorial codes (cf. Ch. 8) [3783]. Egfr is the most widely deployed RTK in ﬂies [1337, 3342, 3348, 3830]. The core RTK transduction cascade was worked out for Sevenless [1048, 1678, 3294, 3671, 4555] and Torso [1117, 1167, 3340] in the early 1990s and was later shown to apply to Egfr [517, 1043, 1337, 1916, 3336]. However, the cascade is not a cassette sensu stricto: depending on the receptor or tissue or stage, certain elements may be replaced by others. For instance, pnt executes Ras-MAPK on/off states in the eye [3162] and wing margin [3025], but not in veins 2–5 [3804, 4191] where the transcription factors are unknown [981], and pnt even acts as an EGFR antagonist during oogenesis [2952]. Cameos of the components involved (or implicated) in EGFR signaling are listed below by abbreviation or full name, with nonenzymatic domains given in braces (cf. App. 1) and sources for molecular-genetic data in brackets. Asterisks mark proteins omitted from Fig. 6.12 because their roles are uncertain (see [1922, 4233, 4286] for additional candidates). The length of the list reﬂects the baroque complexity of this pathway, which seems more like a network: 14-3-3ζ*

{14-3-3}. Scaffold protein that regulates dRaf, etc. [244, 2279, 3638, 3639, 3672].

FIGURE 6.12. The EGFR and Sevenless signaling pathways. See text for proteins, abbreviations, and a summary of how the core chain operates. One cell is sketched with microvilli at top, although ligand contact may take place elsewhere on the surface. Black rectangles = proteins; gray area = cytoplasm (“cyt.”); dashed line below = nuclear membrane. Arrows (activation) and blunt “ ” lines (repression) denote epistatic relations. Noncovalent binding is symbolized by interlocking hooks (cf. key at top). Question marks concern how or whether interactions occur. A “cog” (a.k.a. “adaptor”) is a component that plays a steric (vs. enzymatic) role [441, 1954, 3299, 3676] (e.g., Drk [3185]), while “enz” = enzyme. See also App. 7. Egfr is depicted at the apex but it actually afﬁliates with apicolateral adherens junctions, as do Kek1 and dCbl [1001]. Conversely, the agents that process mSpi (white box at left) [199, 2244, 3385] are shown on the lateral membrane, but they may act apically [3692, 4192] via an ADAM-like protease [4558]. White box at right is a possible module for resetting Ras1 to an off state after signal transmission [3455]. Ras1 resides constitutively on the inner face of the membrane [781, 2653], as do Gap1 [3455], Csw [71], Dos [243], Sos [2137] (ﬂies thus differ from mammals [106, 3485]), and Spry [681, 4340]. The Boss-Sev ligand-receptor complex is shown at right. It plugs into the same downstream circuitry as Egfr (sans Kek1, dShc, or the mSpi add-on) [1081, 3941], although effects of sevGOF and EgfrGOF can differ [1049]. The 7-pass transmembrane ligand Boss (Bride of sevenless) [1732] activates the RTK Sev (Sevenless) [1733, 2319, 2450, 3671, 4359]. Sev differs from Egfr in that its N-terminus is anchored to the membrane to form a big loop [226, 428, 1677] of unknown shape [3136]. Strangely, the entire Boss molecule (ripped from its own membrane?) is internalized by the Sev-expressing cell [601, 2318, 4211]. Sev is concentrated apically [194, 195] and at adherens junctions [4360]. Parochial players in the Sevenless and Torso pathways (e.g., dCdc37 [932]) are omitted. Binding partners in the core chain (listed from surface to DNA) are based on ﬂy studies or vertebrate homologs (vh): Egfr-Drk [3829] (cf. Sev-Drk [3488] ), Drk-Sos [3488] , Sos-Ras1 (vh [402, 4705]), Ras1GTP-dRaf (vh [2913] ), and dRaf-dMEK (vh [898, 2912]). Ancillary binding partners (listed alphabetically, upstream factor ﬁrst): Csw-Dos [243] , dCbl-Spry (not shown) [4738], EgfrCsw (probably indirect based on Sev-Csw [71]), Egfr-dShc (vh [4445]), Gap1-Ras1 (vh [3778, 4044]), Kek1-Egfr [1445], Ksr-dMAPK [2007] , Ksr-dRaf [4285], dMAPK-PTP-ER [2133], dMAPK-Yan [2007], Rap1-dRaf (vh [3057]), Spry-Drk [681], and Spry-Gap1 [681]. Star apparently binds mSpi [1915]. This diagram is adapted from [2131, 3348, 3672, 3782]. It is augmented with data from sources cited above.

180

Argos

Canoe* Clown* Cnk*

Csw dC3G* dCbl

dHsp90* dJun

dMAPK

dMEK

Dos

dRac1* dRacGap* dRaf Drk

dShc E(Elp)24D

Ebi*

IMAGINAL DISCS

(a.k.a. Giant lens or Strawberry) {EGFlike}. Diffusible ligand that inhibits Egfr [1294, 1528, 3183, 3772, 3828]. Binding partner for Ras1 [2748]. Inhibitor that acts downstream of Argos and upstream of Ras1 [4233]. “Connector enhancer of ksr” {PDZ, PH}. Binding partner for dRaf [4288] and essential cofactor for Ras1 [4287]. “Corkscrew” {SH2}. Phosphatase for Dos [70, 3336, 3337]. Likely catalyst for Rap1 [1995]. “dCable” {zinc ﬁnger}. Inhibitor of Egfr [2550, 2821, 3227] and binding partner for Spry [4738]. “Drosophila Heat shock protein 90”. Crutch for dRaf [932, 4447]. {bZip}. Transcription factor regulated by dMAPK [383, 3359, 4381] but surprisingly not by JNK in this instance [2280, 2281, 3593]. “Drosophila Mitogen-Activated Protein Kinase” (a.k.a. Rolled). Kinase regulated by dMEK [332, 517]. “Drosophila MAPK-ERK Kinase”(a.k.a. dSor1 or dMAPKK). Kinase regulated by dRaf [1916, 2700, 4399]. “Daughter of sevenless” {PH}. Adaptor for the receptor complex [243, 1829, 2639, 3489]. GTPase that increases the Ras-dependent activation of dMAPK [4024]. Negative regulator of dRac1 [4024]. Kinase regulated by Ras1 [965, 1050, 2613, 2823, 3491]. “Downstream of receptor kinases” {SH2, SH3}. Adaptor for the receptor complex [3488, 3944]. {PTB, SH2}. Adaptor for the receptor complex [2389, 2623, 4445]. “Enhancer of Ellipse, 24D.” E(Elp)24D binds Egfr (and is phosphorylated by it upon Egfr stimulation), resulting in a diminution of Egfr signaling [4034]. It thus may act like Kek1. {F-box, WD40}. Slimb-like facilitator of protein degradation [1086], which may mediate cross-talk with the Notch pathway.

Egfr Faf*

Gap1

GGT1 Gritz*

Karst*

Kek1

Ksr

Lilli*

Lqf*

Mask*

“Epidermal growth factor receptor.” Receptor for Spi or Vn (in discs) [2573]. “Fat facets.” Protease that cleaves ubiquitin from ubiquitin-conjugated proteins [1931, 4765]. The LOF phenotype of faf resembles that of integral EGFR genes (argos, Gap1, yan) [1932] in the eye (the only disc where faf is needed [1240]), and faf genetically interacts with various components of the EGFR/Sev pathway [1994]. “GTPase-activating protein 1” (a.k.a. Mip or Sextra) {PH}. Antagonist for Ras1 [128, 550, 1399, 3455, 3633]. “Geranylgeranyl transferase-1.” Post-translational modiﬁer of Ras1 [4284]. {EGF-like}. A Spitz-like protein (270 a.a.) that may be a ligand for Egfr [204, 2108, 4558]. Indeed, it may be the missing factor “X” inferred from various system analyses (e.g., see discussion of wing veins below). Cytoskeletal protein (βHeavy -spectrin), whichactsupstreamof Sev.Karstmaymaintain the integrity of adherens junctions for juxtracrine (Boss-Sev) signaling [4296]. “Kekkon1” {Ig-like}. Transmembrane inhibitor of Egfr [1445, 3020]. Possibly inhibits Egfr by sequestering it in inactive heterodimers [77]. “Kinase suppressor of ras” {DEF}. Putative scaffold for a kinase complex [1096, 2007, 4284, 4285] with 14-3-3 proteins [592, 4122, 4772]. Ksr is phosphorylated by dMAPK [592]. “Lilliputian” {HMG} [4250, 4706]. Putative transcription factor that acts downstream of dRaf to regulate the levels of target gene transcription. Lilli also affects the Dpp pathway [3097, 4195]. “Liquid facets.” Endocytosis-associated protein that appears to be a substrate for Faf [594, 1238]. Lqf is needed in the signaling cell, although the identity of the (antineural) signal remains unknown. “Multiple ankyrin repeats single KH domain” {ankyrin, KH, NLS, opa} [4002]. Isolated in a screen for modiﬁers of cswDN , mask encodes a huge (∼4000 a.a.) protein that acts positively in RTK signaling. It is especially needed in R7 photoreceptors [4002], implying that Mask may be more

CHAPTER SIX. THE WING DISC

mSpi

PLCγ

Pnt

PP2A PTP-ER

Rap1 Ras1 RasGAP* Rho

Rin* RnRacGAP* Semang*

Sos Spen*

Spi Sprint*

Spry

involved in Boss-Sev than Egfr transduction. “Membrane-bound Spi.” Spi precursor, which must be cleaved before it can diffuse away from its source cell [3829]. “Phospholipase Cγ” (a.k.a. Small wing) {SH2/SH3/PH}. Activator for Gap1 [3455, 4272]. “Pointed” {Ets}. Transcription factor regulated by dMAPK [516, 745, 2242, 3162, 3804]. “Protein Phosphatase 2A.” Phosphatase for Ras1 and dRaf [2660, 4495, 4554]. “Protein Tyrosine Phosphatase-ERK/ Enhancer of Ras1.” Phosphatase for dMAPK [2133]. (a.k.a. Roughened). Inhibitor of dRaf [1724]. GTPase on/off switch [481, 1273, 3084, 3943]. “Ras GTPase-activating protein” {PH, SH2, SH3}. Inhibitor of Ras1 [1207]. “Rhomboid.” Facilitator for cleavage of mSpi [330] (cf. a paralog that operates during oogenesis [1644]). “Rasputin.” Regulator of a RasGAP other than Gap1 [3300]. (RotundRacGAP). Regulator of the actin cytoskeleton [1642]. Isolated as a modiﬁer of Src42A [4870], semang functions downstream of, or parallel to, Yan [4869]. “Son of sevenless” {PH}. Activator for Ras1 [391, 3635, 3943]. “Split ends” {RNA-binding}. Positively acting component that interacts synergistically with Pnt and antagonistically with Yan [736, 3542]. Spen also inﬂuences the Wg pathway [2554]. “Spitz” {EGF-like}. Diffusible ligand for Egfr [3711, 3829]. “SH2, poly-proline-containing Ras interactor” {SH2, Pro-rich} [4224]. Probable binding partner (and exchange factor?) for Ras1-GTP. “Sprouty” {Cys-rich}. Activator of Gap1 [681, 1665, 2321, 3558] and binding partner for dCbl [4738]. Spry interacts synergistically with Argos [1999].

181

Src42A* Star Svp*

Vn Yan

Part of a branch parallel to dRaf [2612, 4870]. Facilitator for cleavage of mSpi [1782, 1785, 2288, 3385, 3678]. “Seven-up.” Transcription factor ﬁrst ascribed to the Sevenless pathway [1856, 2322, 2888], and later shown to genetically interact with Egfr [264] and frizzled (planar polarity) [1193]. “Vein” {EGF-like, Ig-like}. Diffusible ligand for Egfr [3801]. Transcription factor {Ets, DEF, DEJL} regulated by dMAPK [3467] .

The trunk line of the EGFR pathway appears to work as follows (“vh” indicates data from vertebrate homologs): 1. Binding of ligand causes dimerization and reciprocal Tyr-phosphorylation of the receptor [1814, 3780, 3900, 4556, 4588]. 2. The resulting Tyrp recruits the SH2 domain of Drk [3488, 3785]. 3. Drk’s N-terminal SH3 domain binds the Pro-rich Cterminus of Sos [3488] to alleviate Sos’s self-inhibition [2137]. 4. Sos binds Ras1-GDP, and catalyzes escape of GDP and entry of GTP [391, 3943] (vh [402, 4705]). 5. Ras1-GTP breaks free of Sos (vh [4705]) and recruits dRaf to the cell membrane [167] (vh [1689, 2477, 4128]). 6. Ras1-GTP activates dRaf via dRaf oligomerization (vh [1196, 2620, 2702]), plus an unknown agent [2531]. 7. Ras1 switches back to its inactive state by hydrolyzing GTP to GDP [2633, 2652, 2855, 4043] with or without the help of Gap1 [2880, 3778, 4044], while dRaf phosphorylates dMEK [2700]. 8. dMEK activates dMAPK by phosphorylating it [326, 638, 822, 2700]. 9. dMAPK homodimerizes [823], moves to the nucleus [11, 2206, 2355], and phosphorylates transcription factors that turn various target genes on or off [4252, 4393]. The signal can travel from Steps 1 to 9 in as little as 5 min [1338, 4035]. In the Sevenless pathway, which operates virtually identically up to this point, MAPK phosphorylates dJun [3359], PntP2 (an isoform of Pnt) [516], and Yan [516]. PntP2 is an effector for EGFR signaling in certain contexts

182

but not in wing veins [3804, 4191]. The following additional steps are used in the Sevenless pathway, and comparable ones may operate in the EGFR pathway [670, 3942], although the only disc where Yan is expressed is the eye disc [2392].

[2242, 2952, 3162, 3928, 4775]

10. PntP2 cooperates with dJun [4381] and competes with Yan for cognate Ets-binding sites at the promoters of target genes [516, 1047, 3162, 4393, 4775]. In the R7 photoreceptor equivalence group, the ﬁrst echelon of targets includes phyllopod (phyl) [717, 1047, 1052, 4381]. 11. Phyl forms a complex with Sina (Seven in absentia [671]) and the transcriptional repressor Ttk88 (an isoform of Tramtrack [4773]; cf. Fig. 2.5) [2165, 2529, 4249]. Phyl must be rate-limiting because sinaGOF does not elicit extra R7 cells like phylGOF [717, 1052, 3082]. The complex leads to ubiquitination and degradation of Ttk88 [2529, 4249] via the conjugating enzyme UbcD1 [4249, 4382]. Like Ttk88, Ttk69 (the other isoform) also undergoes signal-dependent degradation [2529] but acts as a transcriptional activator at late stages [2391]. 12. Degradation of Ttk88 alleviates repression of farther downstream genes [2165, 2390, 3082], including prospero [2165, 4775] (a direct target of Yan [670]), other pan-neural genes [4249], and engrailed [4773]. This strategy is reminiscent of the Wg pathway, where genes are controlled by proteolysis of Arm (cf. Fig. 5.6). Among the target genes of the EGFR pathway is Egfr itself, which is downregulated after hours of signaling [4190], thus making cells refractory to further input [681]. Faster negative-feedback loops (via Spry, Kek1, or RasGAP [808, 1445, 3558]) may allow analog responses to graded input [1446, 1527, 1615, 3227, 4557, 4788]. Such dampers favor a rheostat mode, whereas “instant-reset” loops (via PLCγ?) favor an on/off solenoid mode [3455]. One quirk of RTKs is that signaling can be ignited by excess receptor alone (sans ligand) [4556]. If signaling is not extinguished by the instant-reset module, then it will stop due to internalization and degradation of activated receptors [702, 3783, 4482, 4545]. LOF alleles of rho remove parts of veins (mostly distally) [1041, 1368], while mild overexpression (via a hspromoter) induces extra veins [1643, 4191] as well as expanding vein widths. Stronger overexpression (via Gal4 drivers) elicits even more vein material by switching intervein cells to a vein state throughout the wing

IMAGINAL DISCS

[1643, 3126]. Similar LOF and GOF effects are seen for other

genes in the EGFR pathway [517, 1042, 2493, 3558, 4191], including cnk [4288], csw [1237, 3336], dC3G [1995], dMAPK [517, 1043, 1995], dMEK [1916, 2131, 3771], dos [243, 3489], dRac1 [4024], dRaf [1043], drk [1043], dShc [2623], Egfr [181, 814, 3464, 3465, 3484], ksr [1995], Ras1 [1043, 1995, 3470], semang [4870], Sos [1043], spitz (GOF only) [3800, 4604], Star [1643, 1782, 4191], and vn [1366, 3800, 3929], while opposite effects characterize the antagonists argos [3771, 3772, 3800, 3828, 4233], dRacGap [4024], Gap1 [411, 550], kek1 [4604], PLCγ [2669, 4272], and spry [681, 2321, 3558]. The only salient exception is pnt – an EGFR effector in the eye and elsewhere [2242, 2952, 3162], which has no LOF impact on veins [3804, 4191] (although it does affect other wing traits [3928]). This syndrome implies that the EGFR pathway speciﬁes the vein state. Based on heat-pulse studies with a t.s. Egfr allele and a hs-rho transgene, the pathway is required for vein formation at 0–30 h AP, with 6–18 h AP being especially critical [1643]. For years, rho and Star were known to act in the EGFR pathway [4191, 4192], but their functions were obscure. Both genes encode transmembrane proteins (7- or 1-pass [330, 2288], respectively). Recent experiments reveal that Rho cooperates with Star to promote the presentation, cleavage, and release of Spitz [199, 2244, 3385]. Without Rho and Star, Spitz remains bound to the membrane and hence cannot diffuse or activate Egfr on adjacent cells. Neither Vein nor Argos requires such processing because they are directly secreted (sans a transmembrane domain) [3830]. Unfortunately, these new facts do not help solve the problem at hand because Spitz is dispensable for vein development. Wing discs from spitz null embryos develop normally [3928], and spitz null clones do not affect veins [1643, 3025]. Might Rho and Star be assisting other Egfr ligands? The only other ligands known to be expressed in discs are Vn (Egfr activator) and Argos (Egfr inhibitor), but neither of them has a transmembrane domain, so the above talents of Rho and Star seem moot. Indeed, Vn can signal without Rho because overexpressing Vn induces vein tissue in rho-off interveins (Fig. 6.13j) [4604] as effectively as overexpressing Rho [1643]. Rho and Vn must interact somehow, though, because rho LOF vn LOF double mutants exhibit extreme synergy: neither rhove nor vn1 alone deletes more than a few vein sections, but rhove vn1 wings lack all veins except L1 (Fig. 6.13i) [1041, 1366, 2907] due to a failure of vein initiation [320, 3015, 3624]. Some of the major clues about rho and Star are reviewed below:

CHAPTER SIX. THE WING DISC

1. The rho-on state is the earliest indicator of veins [1643, 4191], and rho is the only gene known to be expressed exclusively in veins throughout their development [1643]. Hence, the orthodox view has been that upstream agents funnel through the EGFR pathway [3558] by converging on rho to initiate all veins [1643]. However, the recently revealed rho null phenotype (Fig. 6.13h) [1643] shows that rho is not needed in most of L3 and L4. 2. Rho and Star cooperate in vein development [3385]. When Star (which has little effect relative to rhoGOF ) is overexpressed along with a weak rhoGOF transgene, strong synergy (solid vein areas) occurs at 6–9 h AP, with weaker synergy (extra vein fragments) at 9–24 h AP [1643]. The roles of Rho and Star must differ, however, because Star GOF does not rescue veins removed by rhonull , nor can rho GOF rescue veins removed by Star null (or induce extra veins in a Star null background) [1643]. 3. Rho and Egfr probably act sequentially because no synergy exists between rhoGOF and EgfrGOF transgenes that cause mild increases in vein material [1643]. 4. Both Rho and Star act upstream of Egfr because wings that overexpress rho and Star in the presence of a dominant-negative Egfr (Egfr DN ) show an Egfr DN phenotype, rather than a rho GOF (ubiquitous-vein) trait [1643]. 5. The level of Egfr response can be crudely assessed by an antibody that recognizes the activated (doubly phosphorylated) state of dMAPK [1337, 1338]. This kinase – a key transducer – is encoded by rolled (rl) [332]. Activated Rl (Rl*) is expressed only in rho-on vein cells at 0–24 h AP [1643], except for sporadic Rl*-onrho-off cells next to rho-on stripes [1643]. This congruence suggests that Rho is involved in reception, rather than emission, of a ligand signal [326]. 6. Unlike Egfr LOF clones, which suppress veins cell autonomously [1042, 1043], rho null or Star null clones manifest nonautonomy. In regions of the wing where such clones erase veins, nearby wild-type cells may fail to make part of a vein, and mutant cells at the clone edge can be rescued to make part of a vein [1643]. Both types of nonautonomy imply involvement of Rho and Star in emission (vs. reception) of a vein-inducing signal, thus contradicting point 5 above. In all cases of rho null nonautonomy (point 6), the inﬂuence does not spread beyond a few cell diameters, so

183

the Rho-dependent ligand – whatever its identity – must not diffuse very far. In contrast, Vn’s range must include the whole wing because all veins (save the marginal one) are erased by rho LOF vn LOF . During the main period when the EGFR pathway is needed by veins (6–18 h AP), Vn is expressed in the 3–4 intervein and margin. (After 18 h AP, Vn is expressed in all interveins [3929].) Therefore, Vn, or a secondary messenger, must be diffusing over large (Dpp-scale) distances [1643, 3830]. Conceivably, Vn is activating the pathway everywhere, but only at a basal level consistent with its weak potency [3800]. The level could then be raised above a threshold needed for vein induction by a Rho-dependent mechanism so that veins arise along rho-on stripes. If this “Rho-Vn Booster Model” is correct, then why do the proximal parts of L3 and L4 persist in rho null wings (Fig. 6.13h)? The apparent answer is that these zones experience the greatest dose of Vn because (1) they border iv3–4 where Vn is expressed, and (2) the iv3–4 cells themselves are relatively deaf to Vn because Egfr is downregulated there (Fig. 6.11) [1643, 4604] by Knot [2894] and possibly by Kek1 [3020]. Indeed, the proximal sections of L4 [3929, 4604] and (to a lesser extent) L3 [4189] are preferentially lost in vn LOF mutants. This scenario thus invokes deaf speakers (iv3–4) and mute listeners (rest of the pouch) as per the Deaf-Speakers/Mute-Listeners Trick [328]. Although this logic explains L3 and L4, the Athena Enigma still applies to L2 and L5. In theory, vein-speciﬁc activation of the EGFR pathway could be reinforced by ensuring that inhibitors of the pathway are expressed in interveins. It is thus surprising that the inhibitors Argos (diffusible [1294]) and Sprouty (intracellular [681]) are expressed not in interveins but mainly in veins during this period [2715, 3558]. A similar “Autoinhibition Paradox” has been encountered in other tissues where these inhibitors are deployed [3399, 4081]: why aren’t the cells that produce an inhibitor sensitive to inhibition themselves? Conceivably, their deafness could be due to differences in thresholds or timing of the activator vs. inhibitor [1290, 1528, 1687, 2493, 3347, 4557]. However, neither of these explanations seems to help with wing veins because the pattern and timecourse of argos expression tracks the pathway’s activation too closely (Fig. 6.11). Assuming that the Rho-dependent ligand “X” is expressed coincidently with Argos, the answer may hinge on differences in the diffusion rates of X and Argos [4556]. If Argos diffuses faster than X, then it will venture farther to form a ﬂatter distribution [3347, 3830] – hence

184

IMAGINAL DISCS

1

2

4

5

1

3 4

5

null

+/ Hh Pathway -

+/ Dpp Pathway -

b 71B(pouch)-Gal4:UAS-ptc

+/ EGFR Pathway h rho null

e 756(pouch)-Gal4:

2

1

2

F GO

3

k gbb LOF

Wild-type

Key: LOF

a Wild-type

1

UAS-spalt-related

target gene

3

1

4 3 4

5

f

c Tethered Hh 1

spalt

null

target genes

2

spalt-related

i

null

1

rho LOF vein LOF 1

2/3

3/4 4/5 5 2

d en-Gal4:UAS-hh

g dpp GOF

1

1

2 3

3

4

j

71B(pouch)-Gal4:UAS-vein 1/2/3

4

5

leaving ridges of X “high and dry” above a “sea” of Argos and sharpening the edges of incipient veins. This sort of scenario was devised in the 1970s by Gierer and Meinhardt in their “Activator-Inhibitor Model” [1478, 2802, 2806, 2815, 2818] (a.k.a. Lateral Inhibition [3207]) – a descendant of Turing’s reaction-diffusion scheme [387, 390, 3014, 3208, 4410]. A gene that may encode X has been identiﬁed. The sequence of gritz is 49% identical to spitz [204, 2108, 4558].

But interveins also use the EGFR pathway (at a later time) A drastic change occurs in EGFR signaling at ∼24 h AP. Until then, the pathway is on in future vein cells and off elsewhere, but henceforth the reverse is true [1643, 2715]. During this second phase (24–30 h AP), the EGFR pathway becomes most active in lateral provein bands that ﬂank the veins (Fig. 6.11). Several of the pathway’s

4/5 5

components undergo a similar transition in their expression around this time (viz., argos [2715], Egfr [4190], spitz [1643], and Star [1643]), but rho does not [1643]. Thus, the early congruence between rho-on and Egfr-activated states gives way to a Phase 2 complementarity. The mechanism of this uncoupling is unclear. When the EGFR pathway is forced on in veins (as well as interveins) during Phase 2, L2–L5 fail to form. Evidently, veins must shut off the pathway at ∼24 h AP in order to differentiate. The EGFR pathway is known to switch the allegiance of cells to different fates as a function of time in the egg chamber [2470, 3758] and eye disc (cf. Ch. 7) [1289]. Might a similar switch be happening here so that Egfr implements vein states in Phase 1 and intervein states in Phase 2? No, because the pathway’s chronic LOF phenotypes (vein loss) reﬂect the earlier function only, and suppression of the pathway during Phase 2 only causes a mild increase in vein material [2715]. Evidently, the Phase 2

CHAPTER SIX. THE WING DISC

185

FIGURE 6.13. Dependence of vein pattern on signaling pathways. Black lines or areas mark veins or veinlike regions in all pan-

els, and outer arrows denote vein displacements. For gene abbreviations, see below or legend of Fig. 6.11. The key to terms (above) shows a rheostat dial with LOF and GOF settings on either side of the wild-type level of gene expression. In the panels below, this knob is turned to indicate signal amplitudes for Hh, Dpp, and Vein (or other Egfr ligands). N.B.: When a gene is driven by Gal4-UAS transactivation in an unusual region (stated in parentheses), its transcription is typically higher than in its endogenous region (hence a GOF amplitude effect) [435, 3857]. a. Wild-type wing. b–d. Hh pathway [4478]. This pathway governs veins 3 and 4. b. Overexpressing patched (ptc) in the pouch (via various Gal4 drivers) suppresses L3 and L4 [2992] (but see [4604]). Presumably, the suppression occurs because excess Ptc impedes Hh diffusion [755] and inhibits Hh transduction [2992] , although not severely enough to curtail Dpp-dependent growth. c. A stronger (∼null) effect is obtained when the normal Hh is replaced with a tethered (nondiffusible) construct. This substitution eliminates the 3–4 intervein and collapses L3 onto L4 [4136]. Similar 3–4 fusions are seen with fused LOF [4479] , ptcGOF [2993] , dispatched LOF [572], knot LOF [2894] , and oroshigane LOF [1171] (cf. tout-velu LOF [277]), as well as in transduction-defective fu LOF [3177] or smo null [364] clones that cross from A to P. Milder narrowing of the 3–4 span is seen with EgfrGOF [4604]. d. Overexpressing Hh in the 3–4 intervein region (via en-Gal4:UAS-hh) displaces L3 anteriorly, leaving behind its 3 sensilla campaniformia (circles) [2992]. Widening of the 3–4 intervein is also seen when cholesterol is added to the larval diet [341] – an effect attributable to the cholesterol tail that Hh acquires during its maturation [1960, 1975]. e–g. Dpp pathway [2992]. This pathway governs veins 2 and 5. e. Overexpressing the Dpp target genes spalt or spalt-related throughout the pouch eliminates L2 and L5 (and causes a smaller wing) [981, 984, 2617]. f. Deleting the entire spalt-Complex (i.e., both spalt and spalt-related) in large clones causes fusion of L2 with L3, and L4 with L5. This diagram is a composite of two wings – one with a large A clone and the other with a large P clone (Fig. 2b and c in [986]). Similar effects are seen with dpp LOF [4830] , punt LOF [1674], tkv LOF [1674], and ubiquitously expressed “Supersog” constructs [4830]. g. Overgrowth occurs pervasively, except in the 3–4 intervein, when dpp is overexpressed by (1) using MD-638-Gal4 or 71B-Gal4 to drive UAS-dpp throughout the wing pouch [1537, 2992], (2) using dpp-Gal4 to drive UAS-dpp along the A/P line [2954], or (3) using hs-ﬂp-out to induce scattered dpp-on clones in 2nd instar [4188]. This uncoupling of L2 and L5 from L3 and L4 disproves the simple Dpp Gradient Model (cf. Fig. 6.3) [2992], wherein Dpp speciﬁes all vein positions directly [2954, 4136]. h–j. EGFR pathway [320]. This pathway is instrumental in specifying all veins (except vein 1?), but at a lower level in the control hierarchy than Hh or Dpp (cf. Fig. 6.11). Two Egfr ligands are used redundantly: Vein and a second ligand regulated by rho. Eliminating one or the other (h) represses the pathway, but both must be disabled to shut it off entirely (i). h. When rho function is abolished (by null clones), certain veins (L1 and cross-veins) or fragments (L3 and L4) remain [1643]. Similar remnants are seen in Star null clones [1643]. i. All veins except L1 are absent in rho LOF vein LOF double mutants [1041, 1366, 2907] due to paucity of vein initiation [320, 3015, 3624], and wing shape is altered [1041]. This trait reﬂects extreme synergy because neither of these weak alleles – rhove nor vein1 – has much of an effect by itself [1041, 1368]. Near-total vein loss is also seen with rho LOF argosGOF [3772], rho LOF Egfr LOF [4191], Egfr LOF rolled LOF [1043], Egfr LOF ast LOF [1042], dRafGOF [2715], sproutyGOF [681, 3558], and dpp LOF tkv LOF [980]. j. When 71B-Gal4 drives vein expression in the pouch, the wing is smaller, and 1–3 and 4–5 areas are veinlike [4604]. Similar effects are seen with rhoGOF [3126], rhoGOF StarGOF [1643], spitzGOF [4604], net LOF N LOF [4188], and araGOF [1536]. The immunity of the 3–4 intervein is attributable to its shortage of Egfr [1643, 4604], which, in turn, is enforced by Knot [2894]. This immunity can be overcome at high (Gal4-driven) levels of Rho [1643]. Why the area behind L5 is immune is not known. k. Cross-veins disappear when gbb (glass bottom boat) is repressed [4830], and similar defects are seen with short gastrulationGOF [4830] and tolkin LOF [1233, 3111]. Cross-veins are closer together in approximated LOF , dachs LOF , dachsous LOF , and four jointed LOF (not shown) [4852] . Except for g, which is inferred from preveins in a mature disc (Fig. 2d in [4188]), all sketches (∼ same scale) were traced from photographs of wings in [2072] (b), [4136] (c), [2992] (d), [984] (e), [986] (f), [1366] (h), [4604] (i), [4604] (j), and [4830] (k).

function of the pathway within the interveins involves differentiation more than determination.

Veins 3 and 4 are positioned by the Hh pathway L3 shifts anteriorly when excess Hh is forced into iv3–4 (e.g., by en-Gal4:UAS-hh [320, 2992, 4479]), and it shifts posteriorly when Hh is prevented from entering iv3–4 (e.g., by dispatched LOF [572]). Such displacements indicate that Hh controls the position of L3 (Fig. 6.13) [320]. This inference also draws support from the extra L3-type veins that arise when Hh is overexpressed (with its receptor)

throughout the pouch (via MD-638-Gal4:UAS-hh:UASptc) [2992]. L3 possesses 3 distal sensilla campaniformia [1368, 3234] – the only such stretch receptors in the distal blade [351]. These sensilla are ideally situated to monitor ﬂexion during ﬂight [1045, 1115, 4748] (cf. sensilla on the wing base [840] and haltere [4342]). They come from a proneural stripe along the L3 prevein (cf. Fig. 6.11) [320, 362], although it is unclear how that stripe transforms into 3 PNC spots. When L3 is pushed anteriorly (by en-Gal4:UAS-hh [2992, 4479] or dpp-Gal4:UAS-knot [2894]), the sensilla are often

186

left behind, implying that they are sited independently of L3 (albeit coincidentally) in wild-type ﬂies (cf. other instances of uncoupling [1042, 3624, 4308]). Indeed, the sensilla persist when L3 disappears entirely [320]. L4 often has gaps when Hh diffusion into iv3–4 is impeded (e.g., due to en-Gal4:UAS-ptc) [320], and similar gaps are also seen when vn LOF clones abut the A/P boundary from the A side [1366]. The latter effect is nonautonomous because L4 is in the P compartment, and it suggests a 2-step (signal relay) process consistent with the Rho-Vn Booster Model described above [981, 2852, 2894, 3077]: 1. Hh diffuses anteriorly and activates vn. 2. Vn then diffuses posteriorly to induce L4 [320], which resides just posterior to the A/P compartment boundary [4188]. vn” and “Vn rho”) are known to Both links (“Hh operate in the wing disc [320, 4189, 4604]. The iv3–4 area is kept “vein free” by Knot (a COEdomain transcription factor). Knot’s expression there is induced by Hh [2894, 4479], and knot LOF A clones that abut the A/P line exhibit the same nonautonomy as vn LOF clones (i.e., L4 loss) [3077]. When Knot is misexpressed in other parts of the wing, it is able to suppress veins there as well [2894], so evolution could have used it more broadly but did not (nor is Knot needed anywhere else in discs [3077]). Instead, Bs was granted dominion over all intervein states [2907]. Nevertheless, Knot rules Bs in iv3–4: bs transcription ceases there in knot LOF wings [4479]. Thus, iv3–4 is governed by ≥3 echelons of transcription factors: Hh Ci (zinc ﬁnger) Knot (COE) Bs (MADS) ? intervein. This control system rivals the segmentation gene hierarchy (cf. Fig. 4.2) in the extent of its “middle management.” Knot’s untapped talent as a “vein suppressor” helps explain the perplexing loss of L2 (sic!) in ptcG20 ﬂies [3077, 3372]. Given Ptc’s role as a negative regulator of Hh transduction (cf. Fig. 5.6), this LOF allele should upregulate Hh target genes, including knot, throughout the A region where Ptc normally silences the pathway, and the resulting Knot could be suppressing L2 there. This reasoning is validated by the ptcG20 knot LOF double mutant, whose L2 is completely restored [3077].

Veins 2 and 5 are positioned by the Dpp pathway L2 and L5 disappear when spalt or spalt-related is overexpressed throughout the pouch [984, 986, 2617]. Evidently, these veins rely on on/off expression boundaries that

IMAGINAL DISCS

vanish under these GOF conditions. These paralogs constitute the spalt Complex (Spalt-C). As discussed above, they are turned on by Dpp above a certain threshold [986]. Each of them encodes a zinc-ﬁnger protein [221, 2346] that could directly regulate the transcription of downstream genes. Four of those downstream genes are essential for L2 and L5 (Figs. 6.11 and 6.12) [984]: knirps (a gap gene) and knirps-related comprise the knirps Complex (KniC) [2617, 3653], while araucan (ara) and caupolican (caup) belong to the Iroquois Complex (Iro-C) [1536, 1537]. IroC also includes mirror, but that gene has virtually no expression in veins and instead acts in the alula [2170]. All genes in Kni-C and Iro-C encode transcription factors (zinc-ﬁnger [3060, 3199] or homeobox class [1536], respectively), thus permitting still another echelon of downstream (realizator?) genes. The following facts argue that Spalt-C’s on/off boundaries establish the location of L2 by means of Kni-C: 1. When spalt null clones arise in iv2–3, they can displace the native L2 posteriorly or induce ectopic L2 fragments [984, 986]. The fragments arise just inside the perimeter of spalt null clones [2617, 4188]. 2. When clones deﬁcient for Spalt-C arise in iv4–5, they can displace the native L5 anteriorly or induce ectopic L5 fragments [984, 986]. In this case, the fragments arise at a distance (≤5–10 cell diameters) outside the clone edge [4188]. 3. Knirps is suppressed autonomously wherever a Spalt-Cnull clone overlaps its domain [984], implying “Spalt-C knirps”. Conversely, a high level of SpaltC expression throughout the pouch turns off Kni-C [2617]. Apparently, knirps is only activated at an intermediate Spalt-C level in the same way that genes turn on at thresholds in gradients. Indeed, the knirps-on stripe resides exactly where the native spalt-on band fades out (Fig. 6.11). A “knirps L2” link must exist because (1) knirps is expressed only in provein 2 [984, 2617], (2) Kni-CLOF (associated with deletions or rearrangements) suppresses L2 [2617], and (3) expressing knirps transiently (via heat shock) induces an ectopic vein a few cells ahead of L2 [2617]. Collectively, the data imply that a medium level of Spalt-C knirps L2. In accordance with this linear scheme (essentially a gradient model), knirps can cause the entire dorsal half of the wing to form vein material

CHAPTER SIX. THE WING DISC

when MS1096-Gal4 drives UAS-knirps there [2617]. Because Knirps is normally a repressor [105], it may select the vein state indirectly by repressing the intervein state: Spalt-C knirps bs (intervein state) rho (vein state). Alternatively, the ﬁrst step could be using the DeafSpeakers/Mute-Listeners Trick [328, 2617]. Spalt-C might (1) activate a short-range inducer “X” that diffuses anteriorly to evoke L2 and (2) deafen cells to “X” so that they cannot respond (i.e., Spalt-C X knirps L2). L2 is intriguing because it speciﬁcally acquires bristles in hairy LOF or acGOF mutants [995, 3068, 4189]. Evidently, Hairy normally keeps ac off along L2 (Fig. 6.11) [665], and hairy LOF lets ac rebound enough to support SOP initiation (cf. Ch. 3). The same sort of logic applies to L3, but there is not normally enough Hairy along L3 to keep ac off (thus allowing 3 sensilla), and L3 tends to acquire extra sensilla instead of bristles [362, 2473, 4309]. In some genotypes, the number of bristles along L2 rises to ∼45 [3069]. At such densities, the spacing of bristles becomes strikingly regular. Despite this ability of wing veins to simulate leg bristle rows (cf. L1’s rows), there may not exist any homology because hairy is on between rows in legs, not within them [3193]. L5 also depends on Spalt, but the intermediary agent in this case is Iro-C. Ara and Caup are expressed exclusively in L5 and L3 (Fig. 6.11) [1537], and both veins disappear (L3 partly and L5 completely) when Ara and Caup are suppressed [1536]. The clues to L5’s circuitry are listed below: 1. Ara and Caup are activated ectopically (and autonomously) whenever a Spalt-Cnull clone arises in iv2–3 or iv4–5 [984]. This response implies the link “Spalt-C Iro-C”. As for why iv3–4 is immune, the culprit might be Knot, which is endogenously expressed there: “Knot Iro-C”? 2. Overexpressing Spalt-C throughout the pouch (via 756-Gal4) erases expression of Iro-C in L5 but not in L3 [984]. 3. Overexpressing Ci in the pouch activates Iro-C in the A region (ahead of L3) but not the P region (around L5) [1537]. Apparently, en blocks Iro-C activation: “En {Ci Iro-C}”. En cannot be repressing IroC directly, though, because Iro-C turns on in L5 (squarely in en-on territory). 4. Although Ara is not normally expressed in L4, it can rescue L4 when widely expressed (via 71B-Gal4) in a pouch whose Hh pathway is repressed by excess

187

Ptc (driven by the same Gal4) [1537]. Thus, Ara has an untapped talent as a generic vein inducer. Conceivably, Iro-C is activated within a certain range of Dpp concentrations (Dpp Iro-C) that extends beyond the Spalt-C band by the width of Iro-C provein 5 [984]. The resulting activation of ara and caup there could then evoke a vein via rho [1537]: “Iro-C rho L5”. L3 resembles L5 insofar as its immediate upstream regulator appears to be Iro-C [1536, 1537]. However, L3 cannot be obeying the same “Spalt-C Iro-C” rule as L5 because it lies squarely in the Spalt-C expression zone. (L5 is outside it.) This deﬁance of Spalt-C inhibition explains L3’s persistence (unlike L5) in the face of excess Spalt-C (point 2 above). Nor does L3 depend on Spalt-C (or omb [1626]) in a positive way because Spalt-Cnull clones still make L3 [986]. Nevertheless, L3 does need Dpp (in addition to Hh) because constitutive activation of the Hh pathway in DC0 null dppnull clones cannot sustain Iro-C expression when the clones are too far from the endogenous source of Dpp (at the A/P line) [1537]. (Singly mutant DC0 null clones show no such dependence because they manufacture their own Dpp via “Ci-155 dpp”.) To a ﬁrst approximation, the above facts can be explained by a simple rule (where sound is used as a metaphor for signal strength): Hh is audible AND Dpp is somewhat loud AND you can make a vein, THEN make L3.

IF

This rule accounts for why L3 can be pushed forward by excess Hh (it can still get enough Dpp) but not by excess Dpp (it cannot leave Hh’s diffusion range). However, it fails to accommodate all the available data as the fanciful dialog below shows: skeptic:

If this rule is correct, then why isn’t L3 as wide as the entire Hh gradient? theorist: L3 can’t form if knot is on, and knot turns on when the Hh signal is loud. skeptic: Well, if that’s true and you remove knot, then L3 should widen but it doesn’t! It just moves closer to L4. theorist: Quite right, but no one knows the diffusion range of Hh in the absence of Knot. Maybe Hh can’t get very far without Knot and hence makes L3 closer to the A/P line. skeptic: Rubbish! Hh’s speed depends on Ptc, and knot doesn’t affect ptc! And don’t try

188

invoking Dpp because knot doesn’t affect that either! theorist: Very well, but maybe Knot does more than just enforce an intervein state. Maybe it helps amplify the Hh signal. In its absence the cell would be deafer and would only respond to higher levels of Hh, thus moving L3 closer to the A/P line. skeptic: There’s no evidence for that, and anyway it can’t be true. You’d expect all the cells in a ptc null knot null clone to make vein tissue (because ptc null should activate their Hh pathway similarly, and knot null should let them all make L3), but only the cells in the middle of the clone do so. How do you explain that? theorist: Indeed, the intervein tissue in those clones is perplexing. Maybe the span of L3 tissue is limited by lateral inhibition (mediated by Notch or Argos)? I don’t know. Why don’t we make it an essay question for the grad students? skeptic: Good idea! Maybe they can ﬁgure it out! A related issue concerns symmetry. Given that L2 and L3 are a crude mirror image of L4 and L5 (cf. truly symmetric wings [1647, 3935]), the dpp-on stripe, which supposedly speciﬁes L2 and L5, should coincide with the symmetry plane. Such a superposition may prevail before late-3rd instar [320], but not after the stripe shifts anteriorly along with the en-on edge (Fig. 6.7d) [350]. How can a reference line specify a symmetric pattern if it is off center (closer to L3 than L4)? The solution to this “Registration Riddle” (cf. Gaps Mystery; Fig. 5.1) might be found in en’s role as a selector gene [3074]. If en enables cells on opposite sides of its on/off boundary to interpret their identical Dpp gradients differently, then perhaps it can also make one group deafer to compensate for the displacement. Alternatively, the mismatch might be corrected by a shim strategy that relies on the rainbow of gene expression subzones inside iv3–4 (cf. Fig. 6.3): one or more of those states could be added or subtracted from Dpp inputs to modulate the perceived gradients.

The Dpp pathway later implements the vein state Just as overexpressing rho in the pouch converts almost the whole wing to vein material [1643] (cf. EgfrGOF [3558]), so does overexpressing dpp [980, 1674, 3109] (cf. clones that are dppGOF [4848], tkv GOF [980, 1674], or bs null [2907, 3624]). Con-

IMAGINAL DISCS

versely, reducing dpp activity (in clones or whole wings) suppresses veins [980, 3438, 3850] (cf. tkv LOF and schnurri LOF clones [569] but cf. punt LOF clones [3329]). This late role for dpp in implementing vein identity is separate from its earlier gradient role in specifying vein locations [981]: 1. Prospective vein cells turn on dpp after dpp expression fades from the A/P border. Estimates differ for the time of onset: 6–9 h AP [4831] or 12–16 h AP [980]. 2. Expression of dpp in veins is controlled by a “shortvein” cluster of cis-enhancers [980, 3850, 4056], which is separate from the “disc” cluster that regulates expression at the A/P line [347, 2739]. 3. Expression of dpp in veins is regulated by different upstream circuitry, which must include the EGFR pathway because dRaf dpp [2715]. Indeed, as mentioned above, veins cannot keep dpp on unless they turn off the EGFR pathway at ∼24 h AP [2715]. Because dpp turns on later than rho (or any other known gene) in vein cells, the Dpp pathway may execute the ﬁnal stage in vein cell commitment. Indeed, the period when overexpression of Dpp maximally produces ectopic and thick veins is 16–28 h AP [4831], which is roughly coincident with the period (22–30 h AP) when the Notch pathway is required [1951], but later than the period (6–18 h AP) when the EGFR pathway is essential [1643]. Moreover, dpp must function downstream of the EGFR pathway because dppGOF rescues vein development in rho LOF vn LOF wings (without activating rho) [980]. The diffusion range of Dpp under these conditions is probably minimal because the lateral provein cells that ﬂank each prospective vein express high levels of Thick veins [980]. This high receptor density should “sop up” most of the Dpp before it can travel very far [2457]. Thus, Dpp evidently functions exclusively in an autocrine mode to induce vein determination (and/or differentiation).

A cousin of Dpp (Gbb) fosters the A and P cross-veins In embryos, the product of the short gastrulation (sog) gene is a diffusible Dpp antagonist with a distribution complementary to Dpp [96, 319, 1278]. Interestingly, sog is also expressed in a pattern that is complementary to dpp in pupal wings (i.e., interveins and lateral proveins like tkv; Fig. 6.11) [4831]. However, LOF-GOF studies show that Sog’s role is minor: (1) sog LOF veins are merely irregular in width and alignment, and (2) sogGOF eliminates both

CHAPTER SIX. THE WING DISC

cross-veins. Thus, Sog’s chief duty may be to trim dppon ﬁles of cells. The upstream factors that regulate sog are unknown [2715] but likely include rho [4831]. Another agent that appears to function speciﬁcally in cross-vein development is Glass bottom boat (Gbb) [2203, 4610] – a cousin of Dpp in the TGF-β family [2226, 2734]. LOF mutations in gbb cause the same cross-veinless trait as GOF alterations in sog (Fig. 6.13k) [4830]. Genetic interactions between sog and gbb are much stronger than those between sog and dpp [4830]. An additional crossvein regulator belongs to the same class of BMP1-like metalloproteases as Tolloid (the enzyme that cleaves Sog [771, 1232, 2696, 2990, 3901]) – viz., Tolkin [1233, 3111]. Crossveins are removed by LOF alleles of still other genes [2561] (e.g., crossveinless [469, 4583], crossveinless 2 [854, 3501], and crossveinless-like 6 [2897, 2898]) and by dCul-1GOF [1831]. Wg is speciﬁcally expressed in the cross-veins (but not in L2–L5) at 24–30 h AP [353, 3374], although it is relatively unimportant for their formation [854]. Cross-veins are intriguing geometrically because (1) their orthogonal orientation relative to L2–L5 implies a different reference axis and (2) the anterior cross-vein spans the A/P compartment boundary and hence reﬂects regional coordination [2260]. Ectopic cross-veins form between L2 and L3 in backgrounds that exacerbate vein formation [936, 1431, 3484, 4189, 4228, 4664], and similar conditions foster extra cross-veins at other proximal-distal levels [936, 1431, 3484]. The sensitive period for inducing extra cross-veins via RnRacGAP is 24–30 h AP [1642] (just after the 18–24 h AP window when longitudinal veins can be suppressed), and cross-veins can be eliminated by heat-shocking wild-type pupae at 16–28 h AP [2896]. Cross-veins do not express rho until 25 h AP (vs. L2–L5, which turn rho on by 0 h AP) [854, 4189]. The abiding mystery is why cross-veins normally only form at two ﬁxed sites [2688]. The fact that extra cross-veins appear between L3 and L4 when the iv3–4 span shrinks distally (e.g., due to knot LOF [3077]) suggests that the proximity of adjacent longitudinal veins might be crucial. According to this “Jacob’s Ladder Model” (alluding to sparks that arc between antennae of a Van de Graaff generator), cells would be programmed to make lateral connections wherever the space between longitudinal veins drops below some preset distance at some predetermined time. Alternatively, cross-veins might be speciﬁed by the Wg gradient. These contrasting views are relevant to the larger issue of the extent to which patterning relies on physical (epigenetic) tricks – an old [861, 3063, 3357, 3699, 4306] but ongoing dispute [858, 1557,

189

2154, 2765, 3494]

in “evo-devo” biology

[1489, 1552, 1691, 1876, 3495,

4521].

Vein 1 uses a combination of Dpp and Wg signals The posterior wing margin shows vein markers early in the pupal period but never forms a mature vein [1312]. The anterior margin forms a vein (L1) that resembles the four blade-traversing veins (L2-L5) in histotype and gene expression during the pupal period [1312, 1313, 3015], but it differs from them in (1) its immunity to LOF defects in Egfr [3624] and other EGFR pathway genes [320, 1368] and (2) its failure to express vvl [995]. More important, L1 is perpendicular to the dpp-on stripe along the A/P boundary, whereas all the other veins run parallel to it. The signiﬁcance of this difference can be appreciated by imagining yourself shrunk to the size of a cell. If you were to walk along vein L2, L3, L4, or L5 before evagination, the volume of the perceived Dpp signal would seem constant. In contrast, if you were to stroll anteriorly from the wing tip along L1, the intensity of the Dpp signal would gradually diminish because the Dpp gradient decreases in this direction (cf. Fig. 6.3). The patterns of bristles along L1 deﬁne three basic sections: (1) a “tip” pattern in iv3–4 consisting of long noninnervated hairs plus a few bristles near L3 [1837, 2894, 4479], (2) double row (DR) in iv2–3, and (3) triple row (TR) anterior to vein 2 [1741, 1837] (cf. Fig. 6.7d). The tip pattern is due to the creeping of en expression into this region [1837, 4229], but the latter two sections probably depend instructively on the level of Dpp. Why don’t blade cells respond like L1 cells? It is likely that bristles are conﬁned to the margin because a high level of Wg is needed permissively (i.e., as a digital vs. analog input) to induce achaete in the en-off area as a prerequisite for SOP initiation (cf. Ch. 3) [166, 912, 3689, 3982]. Evidence for this “Instructive-Permissive Model” is listed below: 1. Raising Dpp levels along the whole length of L1. Suffusing the wing pouch with Dpp via MD-638-Gal4 causes DR-like bristles along the whole span of L1 [2992]. 2. Making cells think they hear Dpp more loudly. Raising the perceived level of Dpp in tkv GOF (or mtv LOF ) clones causes cells along L1 to autonomously shift their fates toward more posterior identities – hence making DR bristles in the TR region [1327]. 3. Making cells deafer to Dpp. Reducing the perceived level of Dpp in tkv LOF or sax null clones

190

IMAGINAL DISCS

causes cells along L1 to autonomously shift their fates toward more anterior identities (DR to TR) [3972]. 4. Making cells think they hear Wg more loudly. Increasing the perceived level of Wg throughout the wing pouch (by driving UAS-Dfz2 with 1J3-, 69B-, or 71BGal4) causes bristles to form sporadically throughout the blade [3691], and these extra bristles look like the marginal bristles at their A-P position (DR or TR). A stronger transformation is seen in sgg null clones [351, 3958] (cf. punt LOF clones [3329]). When such clones are far from the wg-on zone, they tend to round up or form vesicles [351, 353, 3603] (cf. similar behavior on the notum [3956] and leg [1039] and its possible signiﬁcance [3046, 3049, 3050]).

These regulators constitute the elusive “prepattern” agents [615, 918, 2890, 2891, 3953] foretold long ago by Stern [4095]. We have only begun to grasp how they control the SOP-making proneural machinery (Fig. 6.14, Table 6.2). Some of the key interactions identiﬁed so far are described below: Link 1:

Link 2:

Macrochaetes are sited by various ‘‘prepattern’’ inputs The heminotum develops as a sort of “island” within the wing disc [157, 793, 1060, 2937, 3104]. It has its own sources for Dpp and Wg, and is insulated from signals that govern the blade and hinge [678, 987, 994, 1074, 2253, 2570]. This island is ruled by an oligarchy of transcription factors or cofactors that evoke macrochaetes at ﬁxed sites: Iro-C

Mad Pan Pannier (Pnr)

U-shaped (Ush)

The Iroquois Complex (140 kb at 69D1-3) encodes 3 homeodomain proteins: Araucan (Ara), Caupolican (Caup), and Mirror (Mirr) [1536, 2170, 2794, 3079]. Iro-CLOF causes an absence of bristles from the sides of the notum – a trait that resembles the “Mohawk” haircut of the Iroquois (a tribe of native Americans) [951, 2520]: hence, the names “Iroquois,” “Araucan” (another tribe), and “Caupolican” (an Araucan hero) [1536]. Terminal effector of the Dpp pathway (cf. App. 6). Terminal effector of the Wg pathway (cf. App. 6). A zinc-ﬁnger transcription factor [1671, 3503, 3504] (a.k.a. dGATAa [4700]) that binds GATA sequences [1380]. A zinc-ﬁnger cofactor that heterodimerizes with Pnr [911, 1671] and represses transcription, probably by recruiting dCtBP [3764].

Link 3:

Link 4:

Link 5:

Dpp (at high levels) ush (cf. analogous regulation in the embryo [127, 2050]). When the Dpp pathway is constitutively activated (in tkv Q253D clones), ush turns on ectopically [3764]. When the pathway is suppressed (in tkv LOF or Mad LOF clones [3764] or in punt LOF or DadGOF discs [4369]), ush turns off within its normal domain. Dpp pnr (cf. the embryo [30, 127, 2050, 4700]). Like Ush, Pnr is expressed ectopically in tkv Q253D clones and extinguished in tkv LOF or Mad LOF clones [3764, 4369]. Dpp (at low levels) wg. When notal cells are “duped” (by tkv LOF , punt LOF , or Mad LOF ) into thinking they are receiving less Dpp than they actually are, the wg-on stripe shifts toward the dpp-on stripe [3763, 3764, 4368], as if the wg-on stripe is “tracking” a certain level of Dpp activity. Likewise, the wg-on stripe moves away from the dpp-on stripe when the perceived activity of Dpp rises (in tkv GOF clones), and it vanishes in dppLOF discs [3763]. High Dpp levels (via ap-Gal4: UAS-dpp or tkv GOF ) exert an opposite effect (Dpp wg) [3763, 4368], probably due to Links 1, 2, and 5: “Dpp {Pnr and Ush} wg”. {Pnr and not Ush} wg. Wg is ectopically expressed when pnr is artiﬁcially turned on beyond its normal ventral edge (by C765Gal4) [1380, 3764], and Wg stops being expressed in its normal (pnr-on/ush-off) stripe when pnr is turned off (by pnr null clones) [3764, 4369]. In pnr LOF ﬂies, the wg-on stripe shifts dorsally [615, 1380, 4369] as it does when the Dpp pathway is repressed (cf. Link 3). {Pnr and Ush} wg. When pnr is turned off (in pnr null clones or pnr LOF ﬂies), wg now turns on in the pnr-on/ush-on (dorsal) region [3764, 4369]. This ability of wg to turn on sans Pnr suggests that Link 3 works at all Dpp levels but is overridden at high

CHAPTER SIX. THE WING DISC

Link 6:

Link 7:

Link 8:

levels by Link 5. However, wg also turns on in this region sans Dpp [4368], so wg may be activated by an unknown gene [3764]. Ush behaves as a negative regulator of wg throughout the pnr-on domain: (1) wg turns on dorsally when ush is turned off there (by ush null clones) [3764], and (2) the wg-on stripe vanishes when ush is broadly overexpressed (by C765-Gal4 [3764] or pnrGal4 [4369]). Pnr AS-C. LOF mutations at these two loci interact synergistically, suggesting that the genes act in the same pathway [3503]. Pnr activates the AS-C directly by binding GATA sites within the cis-enhancer for the dorsocentral (DC) PNC [1380]. This enhancer may loop into contact with the ac and sc promoters via a bridging complex consisting of Pnr, Chip, and Ac/Da or Sc/Da dimers [3504]. Ush AS-C. Expression of ac-lacZ in the DC proneural cluster expands in ush LOF mutant discs and fades under ushGOF conditions [911, 1671]. These effects are evidently mediated by the DC cis-enhancer because the same defects arise when a lacZ reporter is driven by this enhancer alone [911]. Wg AS-C. In wg LOF mutants, ac fails to turn on at the dorsocentral and scutellar PNC sites [3374]. Wg was once thought to bias the competition for SOP fate to the side of the PNC facing the wg-on stripe [3374, 3954]. However, Wg’s role here is only permissive: the site of the dorsocentral PNC is not altered when the distribution of Wg is modiﬁed [1380].

As a consequence of this circuitry, Pnr sets the ventral edge of the wg-on stripe (Link 4), while Ush sets its dorsal edge (Link 5) [1380, 3764]. These same factors also set the V and D boundaries of the DC proneural cluster [1380]. How the A and P limits of this cluster are established remains a mystery, as do various aspects of its temporal dynamics (cf. Fig. 3.3). The other PNCs are even less well understood.

How bristle axons get wired into the CNS is not known Ever since nervous systems were ﬁrst described, the “Position-Projection Mystery” [1455] has been: how do

191

peripheral axons ﬁnd appropriate CNS targets [60, 1392, For bristles on the ﬂy’s notum, the growing axons behave differently depending on their source site [4431, 4432]. Axons from medial bristles send branches to the contralateral side of the thoracic ganglion, while axons from lateral bristles remain ipsilateral [1624]. Within each of these two notal areas, the macrochaete (MC) and microchaete (mC) axons act alike. The areas coincide roughly with Iro-C on or off regions (Fig. 6.14). When sca-Gal4 forces ara or caup to be expressed in all SOPs, the axons of medial mCs now manifest an ipsilateral projection, although the axons of medial MCs retain their identity [1624]. The ability to switch mC-axon projections from one state to another by toggling a regionally expressed homeobox gene implies that sensory neurons give their axons a “birthplace code,” which guides them to predestined targets in the CNS [1451, 1455, 1457, 1460, 3789, 4462]. Indeed, CNS neuroblasts seem to emerge from the embryonic ectoderm in just this way [3981]. One attractive idea is that the encoded addresses use cell-surface markers such as Dscam. This neuronal protein has 38,000 possible isoforms attainable by alternative splicing [700, 1597, 3793] (cf. cadherins [787, 3858, 3870] and neurexins [2874, 4420]). Aside from the notum, axonal pathﬁnding has been examined in legs [3005, 4330] and wings [363, 1463, 4636], but the wiring strategies for these appendages remain obscure [4329]. The best clues so far have come from the antennae [2545] and eyes [1850, 2358, 2708, 3514, 4331] (cf. ocelli [1032, 1350]). For antennae, the determining factor in PNS-CNS wiring is receptor subtype. Insects use various olfactory sensilla to detect odors [2955, 4840, 4841], and most of these sorts of sensilla in ﬂies are found on the 3rd antennal segment [2221, 4469] and maxillary palps [818, 819, 4504, 4506]. Olfactory sensilla fall into three morphological classes: trichoid, basiconic, and coeloconic [1653, 4126, 4127]. Transduction of chemical signals is mediated by 7-pass (∼380 a.a.) transmembrane receptor proteins [3389, 3835]. Receptors are encoded by 57 different genes – 17 of which make no detectable mRNA [4506]. The rules for how the remaining 40 genes operate are simple, as are the rules for how the sensory neurons (∼1000 on each antenna and ∼120 on each palp) connect to the 43 glomeruli in each of the brain’s symmetric antennal lobes [2056, 3626, 4125, 4506]: 2973, 4264]?

1. The gene Or83b is expressed in all neurons, implying that Or83b may serve a scaffolding (vs. ligandbinding) role.

192

IMAGINAL DISCS

A

a

disc

c

V

D

ara-caup

PS

P

Prepattern

mirr

SA

NP

DC p

A P

a PA

dpp

wg

pnr

ush

emc

BarH1

PS SC NP

Pattern

b

ac-sc

SA

a p

PA

heminotum

wg

d

e

pnr ush

Dpp

f CtBP Pnr

ON

Pnr

Ush

wg

Ush

Pnr

OFF

wg

and

CHAPTER SIX. THE WING DISC

2. Each of the other 39 genes is expressed in only a subset of neurons. 3. The number of neurons per subset ranges from 2 to 50. 4. Each neuron expresses only 1 of the 39 speciﬁc genes. Expression of 32 genes is restricted to the antenna, while the other 7 are conﬁned to the palp. 5. Neurons that express the same gene tend to be clus-

193

tered on the antenna or palp. This clustering suggests that receptors might be acquiring “addresses” from a system of positional information, but subtypes are interspersed (i.e., the segregation is imperfect). Thus, a coordinate system sensu stricto is unlikely. 6. Neurons that express the same gene send axons to 1–2 speciﬁc glomeruli on the ipsilateral side and show a symmetric projection contralaterally.

FIGURE 6.14. Landscape of prepattern factors that evoke macrochaetes on the notum (cf. Fig. 3.4).

a. Fate map of a mature right wing disc (simpliﬁed from Fig. 6.1). Dots are actual SOP sites [1925]. Dashed line is the A/P compartment boundary. Directions (cf. compass above): A, anterior; P, posterior; D, dorsal; V, ventral. Within the notal region (dark shading), the future scutellum (posterior end) is demarcated by a thin solid line. b. Heminotum, with macrochaete sites labeled. Abbreviations: “a” or “p” (anterior or posterior members per pair), DC (dorsocentrals), NP (notopleurals), PA (postalars), PS (presutural), SA (supra-alars), SC (scutellars) [2560]. The conﬁguration of SOPs matches the adult pattern, except that scutellar SOPs rearrange (“p” moves posterior to “a”). c. Heminotal domains (shaded) where certain “prepattern genes” are expressed in mature discs. Boundaries are not as sharp as depicted, especially for emc, whose proﬁle of expression has many subtle gradations (cf. Fig. 6.2), including a low background level (not shown). Solid circles are bristle sites affected by LOF mutations (cf. Table 6.2); remaining unﬁlled circles are unaffected. Genetic interactions are indicated by connecting wires ( activation; inhibition). Abbreviations (see references for expression patterns and circuitry): ara-caup (araucan and caupolican) [1536], emc (extramacrochaetae) [913] (see [205] for expression and [1349] for bristle effects), BarH1 (postnotal expression domain not shown) [3763], dpp (decapentaplegic) [3764, 4369], mirr (mirror) [2170], pnr (pannier) [614, 3764, 4369] , ush (u-shaped) [3764, 4369] , wg (wingless) [3764, 4369] . The DC cluster, which forms far from the dpp-on stripe, depends on both Dpp and Wg signals [3373]. The “Dpp wg” link inferred from LOF-GOF studies [3763] is mediated indirectly by Ush [3764, 4369]. Hence, it is omitted, as is a “Wg BarH1” link that only works in part of the notum [3763]. The “ush wg” link requires pnr (not shown; see below) [3764, 4369]. The genes ara and caup are each expressed like the depicted irorF209 (“ara-caup”) enhancer trap in the Iro-C [1536]. In co-stained specimens, their expression appears more complementary to that of pnr than depicted here [614]. Indeed, dorsal pnr LOF clones turn on Iro-C, indicating that “pnr Iro-C” (not shown) [614]. Moreover, such clones sort out from the epithelium, implying a difference in afﬁnity [614]. The overall circuitry suggests that Pnr sets the V limit of the wg-on stripe (excluding the scutellar spot), while Ush sets its D limit [3764]. In fact, the wg-on stripe extends a few cells beyond the pnr-on area in late-3rd instar [614] due to a “Wg wg” link (not shown) that is disabled in wg LOF or dsh LOF discs [4369]. The Gal4 insertion line em462, which exhibits a wg-like A-P stripe [615], is omitted because the patterning effects of its host gene are unknown. d. Dpp gradient that supposedly governs the notal region. Dpp turns on ush and pnr at different thresholds (high vs. low). Dpp also turns on wg, but this activation is overridden where Pnr and Ush overlap. The DC cluster is regulated similarly but lies more dorsally than the wg-on stripe [3373], presumably because the AS-C’s DC cis-enhancer responds to slightly higher levels of Pnr and Ush than wg [1380]. e. “Trident” pattern of notal pigmentation, which is variably detectable in wild-type stocks [2982, 4511]. The pattern may reﬂect control elements at the yellow or ebony locus that rely on the kinds of prepattern agents shown in c [1435, 1442, 1443, 2968, 3055]. This pattern was the subject of Morgan’s earliest (1909) artiﬁcial selection experiments with Drosophila [471, 2283]. The outer stripes of the trident are strikingly prominent in the dipteran genus Zaprionus [615]. See also the beautiful “picture wing” ﬂies of Hawaii [669, 4397]. f. Model for how the “{Pnr and Ush} wg” link is implemented at a molecular level. Circles (center) are a Venn diagram (cf. Fig. 6.10). In the absence of Ush, Pnr is a transcriptional activator (left) [1380]. Ush is thought to mediate repression by recruiting dCtBP (right) [3764] as does Hairy (cf. Fig. 3.12). Direct binding of Pnr and Ush to the wg promoter has not yet been shown [1380]. This diagram is oversimpliﬁed because Pnr and Pnr-Ush probably do not compete for the same DNA sites (but see [4369]): excess Pnr (from C765-Gal4:UAS-pnr) apparently cannot displace Pnr-Ush heterodimers [1380]. Panel c is adapted from [3373, 3764, 3954, 4368]. Registration of the various domains follows [3763] (but see [615]). The thresholds in d summarize results of [3764, 4369], and the model in f is based on data in [1380, 1671, 3764]. N.B.: None of the boundaries in c restricts cell lineage [521, 615, 1373, 3007, 3143] (cf. the Cabaret Metaphor; Ch. 4): the sole constraint on lineage in the heminotum is the A/P boundary [1376, 1377]. Strangely, the zones where Dpp activates target genes are oriented at acute angles relative to the dpp-on stripe itself, perhaps due to modulated diffusion rates (cf. the Arc Scenario; Fig. 5.4). Like ac and sc here (c), the proneural gene atonal is regulated by a zoo of upstream factors in the eye (Fig. 7.9e), but that menagerie is more like a traveling circus since it migrates across the disc.

195

194 TABLE 6.2.* GENES THAT CAN ADD OR DELETE MACROCHAETES ON THE NOTUM BY INDIRECTLY** AFFECTING PNCS OR INHIBITORY FIELDS

Pathway/complex: genes

SC (Scutellars)

DC (Dorsocentrals)

PA (Postalars)

SA (Supra-alars)

NP (Notopleurals)

PS (Presutural)

Hh pathway: hh [6, 2992], cos2 [3373], DC0 [3373], ptc [2992, 3373].

↑ by GOF in pathway (via hh-GOF or ptc-LOF).

↑ by GOF in pathway (via cos2-LOF, DC0-LOF, or ptc-LOF).

—

—

—

—

Dpp pathway: dpp [2954, 2992, 3373, 4831], gbb [4610], punt [3373, 4368], spalt [220, 987, 2455], tkv [4368].

↑ by gbb-LOF. ↓ by GOF in pathway (via dpp-GOF or spalt-GOF).

↓ by LOF in pathway (via punt-LOF). ↑ by GOF in pathway (via dpp-GOF or tkv-GOF) or by gbb-LOF.

↑ by GOF in pathway (via dpp-GOF).

↓ by GOF in pathway (via dpp-GOF or spalt-GOF).

↓ by spalt-LOF (although spalt may be acting independently of dpp here). ↓ by spalt-GOF.

↓ by spalt-GOF.

Wg pathway: wg [734, 880, 987, 3090, 3373, 3374], arm [3373], dally [6], Dfz2 [734], dsh [151, 3373], pan [3591], sgg [3681, 3956].

↓ by LOF in pathway (via wg-LOF or dally-GOF), but not Dfz2-null. ↑ by GOF in pathway (via sgg-LOF, dsh-GOF, or pan-GOF).

↓ by LOF in pathway (via wg-LOF or dally-GOF), but not Dfz2-null. Also by arm-GOF. ↑ by GOF in pathway (via sgg-LOF or pan-GOF).

↓ pPA by LOF in pathway (via wg-LOF). ↓ aPA by GOF in pathway (via wg-GOF). ↑ by GOF in pathway (via pan-GOF).

↓ by GOF in pathway (via wg-GOF).

—

↓ by LOF in pathway (via wg-LOF).

Notch pathway: N [461], fng [2253].

↓ by N-Ax.

↓ by N-Ax. ↓ aDC by fng-GOF.

—

—

—

—

EGFR pathway: Egfr [814, 917, 1042], argos [917], dRaf [917], pointedP1 [917, 4604], Ras1 [917], rho [917], spitz [917], yan [4604] (cf. phyllopod [717, 1052]).

↓ by LOF in pathway (via yan-GOF). ↑ by GOF in pathway (via Egfr-GOF or pointedP1-GOF).

↓ by LOF in pathway (via Egfr-LOF). ↑ by GOF in pathway (via Egfr-GOF) or by dRaf-DN.

↓ pPA by LOF in pathway (via Egfr-LOF). ↑ aPA by Egfr-LOF (but aPA is lost when PNC cells are ﬂooded with Egfr-DN).

↓ by LOF in pathway (via Egfr-LOF). ↑ by GOF in pathway (via Egfr-GOF).

↓ by LOF in pathway (via Egfr-LOF). ↑ by GOF in pathway (via Egfr-GOF).

↓ by LOF in pathway (via Egfr-LOF). ↑ by GOF in pathway (via Egfr-GOF).

Iro-C: ara and caup [951, 987, 1624, 2520], mirr [2170] (cf. Dichaete [3405] and its breakpoints [3079]).

↑ aSC by ara-GOF. ↓ pSC by ara-GOF.

↓ by mirr-LOF (Dichaete effect only?).

↓ by ara-LOF caup-LOF or mirr-LOF.

↓ by ara-LOF caup-LOF. ↓ pSA by mirr-LOF. ↓ by ara-GOF.

↓ by ara-LOF caup-LOF.

↓ by ara-LOF caup-LOF or mirr-LOF. ↑ by ara-GOF.

Bar-C (BarH1 and BarH2) [3763].

↓ by Bar-C-GOF.

↓ by Bar-C-GOF.

↓ by Bar-C-GOF.

↓ by Bar-C-GOF.

↓ pNP by Bar-C-GOF.

↓ by Bar-C-LOF. ↓ by Bar-C-GOF.

pnr [615, 1380, 1671, 1796, 3503, 3504].

↓ by pnr-LOF (but pnr-null clones add bristles).

↓ by pnr-LOF (but pnr-null clones add bristles). ↑ by pnr-GOF.

—

—

—

—

ush [911].

↑ by ush-LOF (but ush-null clones never form bristles here).

↑ by ush-LOF (but ush-null can delete bristles). ↓ by ush-GOF.

—

—

—

—

emc [1349].

↑ aSC by emc-LOF.

↑ by emc-LOF.

↑ by emc-LOF.

↑ by emc-LOF.

↑ by emc-LOF.

↑ by emc-LOF.

*Column headings list notal macrochaetes as pairs (except PS) in a posterior-to-anterior sequence (cf. Fig. 3.4), except that DCs and SCs are abutted because they tend to be affected similarly. Symbols: ↓ (macrochaete loss), ↑ (extra macrochaetes at or near this site). Both bristles per pair are affected alike unless otherwise noted. LOF and GOF denote loss or gain of function. LOF effects are denoted by bold arrows (↓ ↓ or ↑ ) because they are more crucial for understanding the roles of genes in wild-type ﬂies. GOF effects (↓ or ↑) can be misleading (cf. Preface). Gene abbreviations (see App. 6 for Dpp or Wg pathway genes): ara (araucan), caup (caupolican), fng (fringe), mirr (mirror), N-Ax (Abruptex is a type of Notch allele), pnr (pannier), rho (rhomboid), ush (u-shaped). Other abbreviations: DN (dominant negative), PNC (proneural cluster). Mutations in many other genes affect macrochaete number [2561], including some discussed in other ↓ ) [1543]). contexts (e.g., exd null (↓ **“Indirectly” here means that the gene affects SOP initiation via intermediate agents (e.g., Achaete or Notch) that create PNCs or inhibitory ﬁelds (cf. App. 5). This deﬁnition is blurred for the Wg pathway because Dsh (a cog in Wg transduction) affects Notch signaling (effectively short-circuiting the two pathways) by binding Notch [151, 3089, 3689]. The EGFR pathway may fall into a similar gray area because of its interactions with the Notch pathway [1291, 3465, 4898], so Egfr is listed both here and in App. 5. Dichaete (Iro-C) is also dually listed due to uncertainty about its breakpoint effects [3079], and Emc is dually listed because its spatial heterogeneity gives it a prepattern quality [1536, 4368]. The same is true for Hairy, although Hairy does not control MCs [2561]. Effects attributable to early function of Iro-C (i.e., speciﬁcation of notum identity) are not shown [1060].

196

The eye’s CNS projection is also dictated by receptor subtype [795, 1390, 1391, 1539, 1912, 2798], but arrival times of retinal axons in the brain’s optic lobes govern the global order [2357, 3382, 3726] (cf. Ch. 7).

1. Axons from photoreceptors R1–R6 stop growing when they reach the lamina of the optic lobe. 2. Axons from R7 and R8 penetrate deeper into the medulla.

IMAGINAL DISCS

3. Axons that enter the optic lobes ﬁrst innervate medial parts of the lobe, whereas those that enter later innervate successively more lateral domains. Interestingly, the wiring process is directed by familiar morphogens. Ingrowing axons use Hedgehog and Spitz, respectively, to affect proliferation [1933, 1934] or differentiation [2970] of optic lobe neuroblasts [2799, 3726], while the optic lobe itself uses Dpp and Wg for neuropil maturation [2130].

CHAPTER SEVEN

The Eye Disc

Compound eyes have ∼750 facets, with 8 photoreceptors per facet A ﬂy’s face is dominated by its eyes (Fig. 7.1). Each of the two compound eyes is a honeycomb matrix of ∼750 “ommatidial” subunits. Each subunit, in turn, has 8 photoreceptors or “R” cells (R1–R8) for a total of ∼6,000 receptors per eye. At this pixel density, ﬂies see grainier images than humans, who have ≥100,000 receptor cells in the fovea alone [925, 1720, 3415, 4737]. Because ﬂy and human eyes appear to have had a common evolutionary origin [1130, 1407, 2037, 2835, 3093, 3557], the obvious “One Eye or Many? Riddle” is: Did our common ancestor have a simple or a compound eye? If the former, then why/how did insects multiply it [3290]? If the latter, then why/how did chordates reduce it to a solitary remnant [611, 1419, 1848, 3121, 4894]? Of course, there is a third possibility. Our common ancestor might have had only a primitive light detector [968], and we chordate or arthropod descendants then built our own versions of eyes based on the genes that were active at those spots on our face [3382]. The epithelium of the eye disc is a monolayer (as is true for all discs; cf. Ch. 4), but the epithelium of the adult eye is stratiﬁed. Above the bundle of 8 R cells, each adult ommatidium has 4 “cone” cells that secrete the lens (no relation to vertebrate cones). Between the bundles are pigment cells that prevent blurring by absorbing scattered photons: 2 PPCs, 6 SPCs, and 3 TPCs (primary, secondary, and tertiary pigment cells) per ommatidium. SPCs and TPCs are shared between neighboring ommatidia, as are 3 bristles at alternating vertices (Fig. 7.1c), so each ommatidium technically “owns” only 3 SPCs, 1 TPC, and 1 bristle, for a total of 23 unshared cells [3539] =

8 R cells + 4 cone cells + 2 PPCs + 3 SPCs + 1 TPC + 5 bristle cells, although the shaft and socket cells die before the adult stage [3351] (cf. Fig. 2.1 for bristle cells). Photoreceptors R1–R6 share many features, whereas R7 and R8 exhibit certain idiosyncrasies (cf. Table 7.1) [1048]. 1. Rhabdomere location. The rhabdomeres (lightgathering organelles) of R1–R6 form a trapezoid around the rhabdomeres of R7 and R8 [1726, 2354]. Hence, R1–R6 are termed “outer,” while R7 and R8 are termed “inner” photoreceptors. 2. Rhabdomere size. R1–R6 have large rhabdomeres, whereas R7 and R8 have small ones [4715]. Thus, R1– R6 offer high sensitivity, while R7 and R8 provide high acuity [1720, 2044]. 3. Rhabdomere layering. R1–R6 span the height of the epithelium, while the rhabdomere of R7 is stacked atop the rhabdomere of R8. 4. Opsin subtypes. R1–R6 express the rhodopsin Rh1 [2354, 3164, 4895]. R8 may weakly express Rh2 [889]. R7 can express either Rh3 or Rh4 [1272, 3381, 3414]. When an R7 has Rh3, its underlying R8 makes Rh5, and when an R7 has Rh4, its R8 makes Rh6 [779, 780, 3257]. In the absence of R7s, R8s make Rh6, implying that Rh6 is a default state that can be toggled to Rh5 only if the R8 gets an inductive signal (so far unknown) from an Rh3-type R7 [780]. 5. Spectral sensitivity. As a consequence of their opsin composition, R1–R6 are primarily sensitive to light in the blue wavelengths, while R7 senses ultraviolet light and R8 responds to blue-green light [1272, 1726, 2235]. 197

198

IMAGINAL DISCS

medial D

ocelli

fate map

fro

a

MF

antenna ns

P

A V

A1 A2

lateral stalk

A3

P

nerve

D

Ar

V

palpus

eye antennal portion

eva g

in

ion

eye portion frons

ocelli

at

A

ommatidium

badult

eye

light

c pc

antenna A1

B S

S

T

S T B C C C P S S P

A2 A3

C

B

Ar

S

T

3 4 2 5 7 1 6

(Cl-Lbr)

palpus 3 4 2 5 8 1 6

(Lab)

To CNS 6. Depth of axonal penetration into the brain’s optic lobe. The axons of R1–R6 terminate in the outer layer (lamina) of the optic lobe [2277, 3407, 4261], while those of R7 and R8 penetrate deeper to reach the medulla [1390, 1539, 1912, 2357, 2798, 4215].

The rhabdomeres of R1–R6 form a trapezoid [1305, and the trapezoids point in opposite directions in the dorsal (D) and ventral (V) halves of the eye [1059]. Thus, the eye is endowed with a D/V plane of mirror symmetry that deﬁnes its “equator” (Fig. 7.2). This 3877],

CHAPTER SEVEN. THE EYE DISC

199

FIGURE 7.1. The eye disc, its adult derivatives, and the cellular components of an ommatidium.

a, b. Fate map (abridged) of an eye-antenna disc (a) and its cuticular derivatives (b). Abbreviations: A1–A3 (antennal segments 1–3), Ar (arista), MF (morphogenetic furrow, black bar; arrow shows direction of movement). The median hemi-ocellus (half oval) fuses with its counterpart from the other eye disc (not shown) [2864]. a. A left eye-antenna disc at maturity. In size and shape, the eye part crudely resembles a wing pouch, and the antenna part looks like a half-size leg disc. Dots are photoreceptor clusters. The nerve exits through the “optic stalk” (unlabeled). The disc’s hind half is cup shaped (concave-side down) with two curled ﬂaps (dashed lines on underside). The antennal part (medium shading) telescopes toward the viewer during eversion. Directions (compass at right) are prospective (a) or actual (b) axes of the adult. The vectors are confusing because eye discs rotate ∼180◦ after they arise in the embryo, hence inverting their A-P and D-V axes [4146]. Thus, the P compartment (≈ en-on domain) winds up anterior [2931], and the original D compartment winds up ventral (compare the fringe-on domains in Figs. 6.8 and 7.4) and is conventionally called the “V” compartment (as shown in the inset box). This inversion explains (1) why the dpp-on and wg-on sectors of the antenna are upside-down relative to those of the leg (compare Figs. 7.3 and 5.8) [2631, 4277] and perhaps also (2) why disabling the Dpp pathway (via dpp LOF or Mad LOF ) removes the D half of the leg but the V half of the eye [723, 930, 1812, 4648] (but see [2752]). N.B.: The antennal D-V axis (mislabeled in [1037]) is deﬁned by Wg and Dpp independently of the global D and V compartments. The A/P boundary is established by late-2nd instar (∼72 h AEL) [2931, 2933], and the same is true for antennae transformed into legs [1635, 2926, 2933], whereas the D/V compartment boundary arises in early 1st instar [189]. These times contrast with the blastodermal onset of A/P restrictions in thoracic discs [4651], but they resemble the timing for the D/V line in the wing disc [354]. See Figure 6.9 for the origin of the eye vs. antenna dichotomy. b. Left half of a head (frontal aspect). The frons manifests parallel grooves like a human ﬁngerprint [1224, 1866]. Unlike the compound eyes, ocelli do not focus images, but rather may detect moving shadows [293]. The arista’s branches may arise from hairs (vs. bristles) [3362]. Unlike the 3rd antennal segment, which senses odors [2221], the arista appears to sense temperature and humidity [1260]. Blank areas show where structures made by other discs would insert: “Cl-Lbr” (clypeus and labrum) from clypeolabral disc and “Lab” (labellum) from labial disc. Interommatidial bristles are omitted. Reshaping during evagination is complicated (not shown) [1311, 1777, 2864, 2865, 3516]. c. One facet from the lattice is shown in frontal view (left). At right is a side view of the entire conical ommatidium – a simple eye [2909, 3230]. Cross-sections are sketched (left) at 3 levels (dashed lines). Abbreviations: B (bristle), C (cone cell; no relation to “cone” photoreceptors in vertebrates); P, S, T are primary, secondary, and tertiary pigment cells; 1–8 are photoreceptors R1–R8. Nuclei are omitted. In the upper section, 4 C cells are embraced by 2 P cells, which are bordered by 6 S cells, with 3 B and 3 T cells at alternating vertices. Adjacent ommatidia share S, B, and T cells (cf. Fig. 7.2). Light is refracted by the cornea (dark shading) and underlying pseudocone (light shading) [1720, 4364]. The cornea is a cuticular secretion of the cone and primary pigment cells, while the pseudocone (pc) is a gel secreted by cone cells alone [602, 2034]. Photons are then transduced by the 8 rhabdomeres (black bars at right = numbered circles at left). Rhabdomeres are compact arrays of photosensitive microvilli [715, 844, 1817, 2323, 3568, 4458] on R1–R8 cells (shaded; intercellular spaces are exaggerated) [1270, 2594, 3350, 4270]. R7’s rhabdomere lies atop R8’s. Both are smaller than the other six, which nearly span the height of the ommatidium. Note (lower sections) that the cytoplasmic stalks (and hence rhabdomere gratings) of R7 and R8 are orthogonal [1318, 4355] – a cute trick for detecting polarized light [1720, 4398]. The transduction cascade culminates in electrical signals [374, 2908, 2909, 3229] that are sent to the CNS via axons (below) [1390, 2801]. Stray photons that might ricochet to adjacent ommatidia (and hence degrade the image) are absorbed by the intervening pigment cells [1048]. These pigment cells thus serve as insulating walls [3369]. Panel a is redrawn from [1777], with details added for ocelli [3663] and photoreceptors (Fig. 1b of [724]), b is adapted from [1777], with territories ascribed to different cephalic discs as per [1408], and c is simpliﬁed from [602, 1048, 1239, 1726, 4715]. See [3540] for a 3-dimensional rendition.

conﬁguration, plus the wiring pattern of axons into the lamina, enhances visual acuity and makes the equator a sensitive “fovea” [433, 931, 1720, 2234, 2800]. The logic underlying this arrangement is a masterpiece of micro-optics and microcircuitry. The visual axes of the rhabdomeres in a single ommatidium diverge by exactly the same amount as the optical axes of neighboring ommatidia. Consequently, the axis of each rhabdomere actually coincides with those of six other rhabdomeres, each in a different ommatidium. The axons of each set of seven optically aligned rhabdomeres (in seven different ommatidia) converge onto a retinotopic unit in the first optic ganglion, the lamina. Here the signals of the six R1--6 cells are summed synaptically, while

the axons of R7 and R8 bypass the synapse and project to the second optic ganglion, the medulla. Therefore, for each point in space, light is collected through six different facets thus greatly enhancing the intensity of the retinal image, but without compromising spatial acuity. This is known as the neural superposition principle. [1721] An intriguing exception to the rule that one point illuminates seven rhabdomeres is found at the so-called equator of the eye, about which the characteristic rhabdomere patterns are found to be mirror-symmetrical. In this region, the axes of eight or nine rhabdomeres in eight or nine ommatidia coincide. Remarkably, the projection pattern of the retinula cell axons follows this quite faithfully into the cartridges, and in the lamina the corresponding cartridges have crowns of

200

IMAGINAL DISCS

Key:

D A

marked V clone unmarked background

B

P

S

S

T S B S P

3 4 2 5 1 6

T P S B 7

S

D

T

V

8

D V

b D V

leg clones

Polarity: D-type V-type

a

c A P

D

a

d

b

V

CHAPTER SEVEN. THE EYE DISC

201

seven or eight receptor axons. Each neuro-ommatidium in this equatorial stripe can thus be expected to have an increased absolute sensitivity. [1720]

Francis Bernard reported histological evidence (albeit circumstantial) for such a clonal mechanism in insect eyes [298]. “La r´etinule” is the photoreceptor array: R1–R8.

Unlike the bristle, the ommatidium is not a clone

Sur un materiel ´ favorable, a` savoir la nymphe de Formicina ﬂava , j’ai pu retracer la filiation cellulaire des el ´ ements ´ de l’ommatidie: il y a, des ` les premieres ` divisions de l’ebauche ´ hypodermique, separation ´ tres ` precoce ´ entre un groupe nucleaire ´ interne et un groupe externe. La serie ´ interne conduit a` la retinule ´ et aux cellules pigmentaires qui l’entourent, la serie ´ externe conduit, plus tardivement, aux cellules corneennes ´ et cristalliniennes. Ces faits n’apparaissent pas dans le texte des prec ´ edents ´ auteurs, bien que les figures de Moroff (1912), relatives a` l’ebauche ´ optique de Palaemon, contiennent implicitement les memes ˆ series ´ nucleaires, ´ dont la gen ´ eralit ´ e´ rec¸oit ainsi un debut ´ de confirmation. [298]

A priori, the crystalline perfection of the ommatidial lattice and the geometric precision of its attendant trapezoids seem to demand an equally regimented process of assembly. One obvious solution would be a cell-lineage mechanism. An ommatidial “mother cell” could undergo a ﬁxed number of differentiative mitoses to produce each of the needed cell types, just as bristle SOPs divide to make the 5 cells of the bristle organ (cf. Fig. 2.1). It was therefore not surprising when, in 1937,

FIGURE 7.2. Lineage restrictions at the eye’s equator. Until recently, the eye disc was thought to lack a D/V compartment boundary because clones can cross the equator. However, such transgressions are trivial (a, b), and similar trespassing occurs at bona ﬁde compartment lines in other discs (c, d). In both cases, the “noise” is attributable to patterning events that are superimposed upon – but only loosely linked to – the boundary. The key (top) shows (left to right): clone vs. background (black/white) markings; axes (D-V, dorsal-ventral; A-P, anterior-posterior); ommatidial template (B = 5-cell bristle organ; P, S, T = primary, secondary, and tertiary pigment cells; 1–8 = photoreceptor R1–R8 cells); conjectured locations of clones (enlarged in a and b) within their respective eyes (ovals); and trapezoid orientations in the D vs. V halves of the eye. Within the lattice, the ommatidial “repeat unit” has 23 cells, counting each bristle as 5 cells and excluding the cells that each ommatidium shares with its neighbors [3539]. a, b. Clones of cells (black) that are homozygous for white LOF as a result of somatic recombination induced by X-rays in late 1st-instar heterozygotes [3539] when the eye rudiment has ∼20 cells. Because an adult eye has ∼750 ommatidia and only one of the two cross-over segregants becomes homozygous [1695], a single cell that undergoes recombination should leave marked descendants in 750 × 1/20 × 1/2 ≈ 20 ommatidia. Indeed, each of these clones covers ∼20 ommatidia, but most are mosaic. The mosaicism shows that the ommatidium is not a clone. In the living ﬂy, each clone was a ragged white stripe across an otherwise red (left) eye (cf. key). Except for bristles (shaded), all the cell types shown are scorable phenotypically. The equator (D/V line at left) is deﬁned by the symmetry planes of the columns (13 columns per eye are shown). The R1–R8 trapezoids in each column always point dorsally above a certain level and ventrally below it [2794], so that no D-type ommatidia ever get “stranded” in the V half or vice versa [4797]. The equator zigzags through the lattice, jogging by one ommatidium from column to column. Normally it alternates – jogging up then down (sawtooth mode) – but in places it jogs one or two rows in the same direction (staircase mode) before returning to the baseline [2794, 3539, 4715]. a. Clone that evidently originated in the D half but which includes a few cells in 3 adjacent V-type ommatidia (arrows). b. Clone that must have arisen in the V half but which includes some cells in 4 D-type ommatidia (arrows). All but one of these 7 ommatidia (rightmost arrow in b) have a majority of their cells from the “home” compartment. This same trend is seen in Minute mosaics where the marked clones (induced at various ages) have a growth advantage [633]. c, d. Clones (black) marked with yellow LOF on two left 2nd-leg basitarsi (proximal-distal axis is left to right). Only one row of 11 bristles (circles) – row 1 – is depicted. This row resides at the A/P compartment boundary. Its bristles can come from either compartment, depending on the individual leg. c. All bristles, except the second one, must have arisen from P cells because they are embraced by a clone of P provenance. d. Several of these same bristles must have arisen from A cells because they are in a clone of A provenance. Such vagaries probably reﬂect the stochastic way that SOPs are selected in proneural ﬁelds (cf. Fig. 3.9). Panels a and b are redrawn from [3539]; panels c and d are redrawn from [1800] (6th leg in his Fig. 3b and 4th leg in Fig. 3a). In the key, the locations of the clones along the A-P axis (arbitrarily sited here) were not actually reported in the original paper. In c and d, the contour of the clone boundary through the background epidermis (drawn straight) is not actually known because the yellow LOF mutation does not affect ordinary cuticle. N.B.: Clone edges (a, b) do not look this ragged in 3rd-instar discs, especially near the D/V border [696]. The choppiness seen here in the adult is due, at least in part, to 90◦ rotation of ommatidial clusters in late-3rd instar (cf. Fig. 7.5a–c), which must detach closely related cells from one another at cluster perimeters by breaking junctions. There is no A/P compartment boundary in the eye itself, which lies entirely in the A region (cf. Fig. 7.1) [4146].

202

Bernard’s “Ommatidial Lineage Hypothesis” prevailed until 1973, when it was disproven in Drosophila by Seymour Benzer and his collaborators [292]. Benzer had been a hero in the Phage Genetics Era of molecular biology [291, 610, 1884, 4083, 4560], and he would go on to become a patriarch of modern ﬂy genetics [4578]. In a popular article for Scientiﬁc American (1973), Benzer sketched their essential ﬁndings. Their refutation of the lineage model was based on somatically marked clones – a lineage-tracing method that is more accurate than histology alone. Are the eight photoreceptor cells derived from one cell that undergoes three divisions to produce eight, or do cells come together to form the group irrespective of their lineage? This can be tested by examining the eyes of flies, mosaic for the white gene, in which the mosaic dividing line passes through the eye. . . . The result is clear: a single ommatidium can contain a mixture of receptor cells of both genotypes. This proves that the eight cells cannot be derived from a single ancestral cell but have become associated in their special group of eight irrespective of lineage. The same conclusion applies to the other cells in each ommatidium, such as the normally heavily pigmented cells that surround the receptors. [292]

A full account of the data came three years later in a Developmental Biology paper with the catchy title, “Development of the Drosophila retina, a neurocrystalline lattice” [3539]. Authored by Donald Ready, Thomas Hanson, and Benzer, this article was remarkable not only for its rejection of the clonal model [2128, 3233] but also for its pithy questions about eye development in general. It launched a brute-force mutational dissection of eye patterning that is arguably the greatest success story in modern genetics [293, 1048, 4471, 4715]. Despite its seminal contributions, however, the Ready et al. article had one unfortunate side effect. Like Bernard’s paper it disseminated a myth of its own – viz., that the eye lacks D and V compartments (italics are author’s) [3079]. The shape of a marked clone . . . depends somewhat upon its position in the eye. The dorsal or ventral edge of a patch often runs horizontally near the middle of the eye. This suggests the possibility that the descendants of early cells tend to populate either the dorsal or ventral halves of the eye (Becker, 1957). . . . [However,] while the cells of a white clone tend, by and large, to form a continuous patch, some marked cells lie removed from the main body of the clone. Such outlying cells are rarely more than one ommatidium removed from the main patch. . . . Thus, although the equator often lies near a clonal boundary, it is not determined by such a boundary. [3539]

IMAGINAL DISCS

The history of this topic deserves scrutiny because it shows how paradigms can shift with the tides of opinion, not just with the facts on which the opinions are based.

The eye has D and V compartments (despite doubts to the contrary) The equator was discovered by Wilhelm Dietrich in 1909 [1059]. In 1957, Hans Becker found that marked clones tend to respect the equator [260], and a similar conclusion was reached in 1967 by William Baker [188]. The exact path of the restriction line was uncertain, however, because neither Becker nor Baker assessed the orientations of the trapezoids [2128]. When A/P and D/V compartments were revealed in the wing disc in 1973 [1376], the eye’s previously known D/V restriction boundary acquired, at least potentially, a new meaning. The suspicion was that the eye’s D/V line might be acting like the wing’s D/V border. This “D/V Compartment Hypothesis” was appealing because it offered the prospect of a universal coordinate system for discs. In 1978, the “Minute technique” [2935] was used to assess whether this line behaves like a compartment boundary under “pressure” from overgrowing clones. Indeed, it does [189]. That is, it is not transgressed by faster-growing Minute+ clones in a Minute LOF background. More important, clonal boundaries in the Minute mosaics were found to track the equator closely in the trapezoid array [633, 1080]. From one eye to the next, the discrepancy is typically no more than ±1 row of ommatidia (Fig. 7.2). That is, D clones “spill over” the equator by no more than one V-pointing trapezoid (and vice versa). This degree of “noise” might seem negligible, but Ready et al. [3539] and others [1804, 1869, 2128, 4715] viewed any discrepancy as a sufﬁcient reason to reject the hypothesis. At that time, this attitude seemed reasonable because all the then-known compartment lines were sharp and inviolate. The skepticism of these researchers was based on other facts as well [696]. The full list of attacks on the D/V Compartment Hypothesis is given below: 1. “Trespass” Objection. The ability of marked clones to cross the equator (even slightly) argues against the D/V line being a canonical compartment boundary. Shouldn’t the edges of such clones be in perfect register with the equator [3539]? The lack of a D/V

CHAPTER SEVEN. THE EYE DISC

boundary in the leg [1800, 4076] lent added plausibility to the notion that the eye might also lack such a boundary [1807]. 2. “Mosaicism” Objection. The ability of marked clones to meander through the ommatidial array without respecting the boundaries between ommatidia [3539] ruled out a clonal mechanism (see above). It also seemed to preclude any strict D-vs.-V dichotomy. If ommatidial polarity is due to a difference in gene expression, then shouldn’t ommatidia behave as units? 3. “Selector Gene” Objection. No mutations were known that convert one half of the eye into the other, although such a phenotype would be hard to detect in an ordinary mutant screen. Shouldn’t the eye possess a selector gene for the D-V axis [633]? The Trespass Objection was undermined in 1979, when a similar transgression was found at the A/P boundary of the leg [1800, 2449]: the bristles of tarsal row 1 can be embraced by either A or P clones (Fig. 7.2c, d). This variability from one ﬂy to the next is attributable to the stochastic way that SOPs are selected. The proneural ﬁeld for row 1 straddles the A/P line, so its SOPs can arise on either side (cf. Fig. 3.9). If bristle rows “don’t care” about the compartmental provenance of their cells, then maybe ommatidia don’t either. In that case, ommatidia would arise from “ﬁelds” that overlap the D/V line, and D/V-mixed ommatidia would still somehow manage to point dorsally or ventrally, rather than freezing at an intermediate angle. The Mosaicism Objection has withered as we have learned more about how ommatidial polarity is controlled. We now know that ommatidia do not orient themselves based on the D-vs.-V states of their component cells. If that were true, then mosaic trapezoids (with foreign cells from across the equator) should point at intermediate angles, but they obviously do not (Fig. 7.2). Rather, polarity is superimposed nonautonomously on ommatidia by a signal [4366] that diffuses from a line between mirror-on and mirror-off cells. It is thus possible for a trapezoid to straddle the mirror-on/off line and still adopt a purely D or V orientation. The way in which this happens is discussed shortly. The most serious criticism of the D/V Compartment Hypothesis was always the lack of a D/V selector gene in the eye (like apterous in the wing; cf. Ch. 6) [625]. One early indication that such a gene might exist came from Ophthalmoptera mutants. In these hideous ﬂies, the

203

eyes are replaced by wings, and the margins of the wings grow out from the equator [3439] – precisely the site expected if the D and V parts of the eye and wing discs are homologous [696]. Another hint came from transplantation experiments. When fragments of the eye disc were transplanted to host larvae, an equator formed only from pieces that contained both D and V tissue [629]. Pieces with only D or V tissue made ommatidia with disordered polarity. Evidently, ommatidial polarity depends on the equator, which, in turn, depends on an interaction between D and V tissues.

The Iroquois Complex controls D-V polarity via Fringe and Notch As mentioned in Chapter 6, the Iroquois Complex (IroC) has 3 homeobox genes: araucan (ara), caupolican (caup), and mirror (mirr). Their DNA sequences were described in 1996 (ara and caup) [1536] and 1997 (mirr) [2794]. All three genes are transcribed in the D half of the eye rudiment [483, 2170, 2794, 4210], beginning as early as late1st/early-2nd instar (Fig. 7.3) [696, 2170]. Intriguingly, the on/off boundary of Iro-C expression is a straight line that closely tracks the equator (± a few cell diameters) [696, 778, 2794]. In 1999, Florencia Cavodeassi et al. (in Madrid and Cambridge) showed that the Iro-C exhibits many of the properties expected for a D/V selector gene [696]. This evidence swept away the last remaining objection to the D/V Compartment Hypothesis. Hence, the eye does develop like a wing along its D-V axis (Fig. 7.4) [359]. This “Iroquois Epiphany” of 1999 has led to a rethinking of the rules for large-scale patterning in the eye. 1. Dorsal (but not ventral) Iro-Cnull clones induce an ectopic equator at their edge. Wild-type trapezoids as far as 7 ommatidial rows away from the extra equator are repolarized (nonautonomously) so that they point away from this new line (Fig. 7.4) [696]. Weaker effects are seen near mirr LOF D clones (which remain ara+ caup+ ) [2794, 4797], as well as with fng LOF V clones [774, 1080, 3258], N GOF , Dl GOF , or Ser GOF clones (see below) [3258]. 2. Ectopic (GOF) expression of ara, caup, or mirr in a subset of the V region causes similar repolarization [696, 2794]. 3. Dorsal (but not ventral) Iro-Cnull clones have round shapes with smooth edges [696, 697], as do mirrLOF D clones [4797]. This trait mimics null clones for D/V (ap) and A/P (en) selector genes in the wing disc

204

IMAGINAL DISCS

gro

Start

w

en

mirr

AND

hh

Egfr ? ?

w

gro

N ?

ci

wg

fng

toy

ey pnr

eya

so

omb

m

dac

w

gro

hairy

dpp

fj

D AN

hth

Dll

WR122

AND spalt

ss

medial D P

A V

lateral

(cf. Ch. 6), and is presumably due to differences in cell afﬁnities [2792]. 4. Iro-Cnull (but not wild-type) clones can cross the equator from D to V [696], again implying an effect on regional afﬁnities. 5. A selector role is hard to prove in the eye because its D and V ommatidia look identical except for ori-

entation. Stronger proof comes from the head capsule where (1) GOF misexpression of caup induces Dtype bristles in the V area [1080] and (2) Iro-Cnull (LOF) clones manifest V-type structures in the D area [697]. If Iro-C endows cells with D-vs.-V identities and those identities dictate ommatidial angles, then Iro-Cnull

CHAPTER SEVEN. THE EYE DISC

205

FIGURE 7.3. Patterns of gene expression in the eye-antenna disc during 3rd instar. Smaller drawings indicate earlier stages. Black areas chart mRNA (transcribed from the endogenous gene or a reporter). Shades of gray denote degrees of expression. Directions are shown in the compass at the lower right: A-P (anterior-posterior) and D-V (dorsal-ventral) refer to prospective axes of the adult, whereas “medial” and “lateral” denote directions within the larval body. Genes (“DO” = details omitted; “ambiguous” means that conﬂicting reports have here been summed into a maximaldomain composite): ci (cubitus interruptus) [1135], dac (dachshund; early-3rd instar) [2353] , Dll (Distal-less) [1085, 3242] , dpp (decapentaplegic; above is early-3rd instar [930] ) [2004] , en (engrailed) [4168] , ey (eyeless; early-3rd instar) [930] , eya (eyes absent; early-3rd instar) [2353] , fj (four jointed; antennal expression is approximate) [4851] , fng (fringe; ambiguous) [696] , hairy [1616] , hh (hedgehog; above is 2nd instar [1082]) [406] , hth (homothorax) [1085, 3380] , N (Notch; 2nd instar) [2353], mβ (E(spl)mβ) [871] (but see [991]), mirr (mirror; ambiguous) [4797] , omb (optomotor-blind) [4849], pnr (pannier; mid-3rd instar) [2752], so (sine oculis; early-3rd instar) [2353] , spalt [1085] , ss (spineless) [1119], toy (twin of eyeless; 2nd instar) [2353] , wg (wingless; above is early-2nd instar [696]) [3762] , and the enhancer trap WR122-lacZ [1781]. Genes omitted (expression patterns is in parentheses): araucan and caupolican (≈ mirr) [3380] , extradenticle (Exd is nuclear wherever Hth is present) [677], orthodenticle (ocellar region and ommatidia) [3663] , vein (just posterior to the Hh ocellar spot) [80]. Annular expression domains of BarH1 (not shown) and dachshund in the antenna appear identical to corresponding tarsal domains (Fig. 5.11) [3762]. Links ( activation; inhibition) indicate genetic interactions; “and” means that both inputs are needed to activate the target gene; dashed lines denote weaker effects. The ﬂow of control begins at upper left in 2nd instar. Whether Egfr inhibits toy directly or via N is unknown [2353]. Egfr is probably activated in the antenna (cf. Fig. 6.9) [2353, 2362, 3382]. The antenna resembles the leg (cf. Fig. 5.8) insofar as “En Hh {dpp or wg}” [1037]. For the eye’s D-V axis, polarity is established by mirr (upper right; cf. Fig. 7.4). Wg may not actually inhibit dpp at the level of transcription [4390]. A “dac eya” link detected elsewhere does not work in the eye [554]. The “and” logic for the early eye genes (middle left) has been omitted for clarity. For an exegesis of this nexus, see [1787]. Circuitry of these genes is based on [744, 930, 1780]. For ideas about how this network evolved, see [1419, 2034, 3895]. For enhancer-trap patterns, see [315, 3388, 4210] and FlyView (ﬂyview.uni-muenster.de) [2027], and for expression of hh, wg, and dpp in the peripodial membrane, see [773]. N.B.: In comparing these expression patterns with those in the leg and wing discs (Figs. 5.8 and 6.2), it is important to recall that the eye disc rotates ∼180◦ after emerging from the embryonic ectoderm [4146], thereby inverting its D-V axis relative to the D-V axes of the thoracic discs (cf. Fig. 4.3a).

clones in the D half of the eye should ﬂip trapezoids from D- to V-type orientation. In fact, the trapezoids within such clones retain a D-type posture (Fig. 7.4), and inversions only occur nearby (point 1 above). This result suggests that ommatidia obey the following two rules: Equator Rule:

Polarity Rule:

If you reside where Iro-C-on and -off cells meet, then become an “equator-type” cell (nature unknown) and emit a diffusible signal. If equator-type cells are nearby, then point your trapezoid away from them.

How might such rules be implemented? Conceivably, the eye’s D-V axis operates like the wing’s, with the Iro-C substituting for apterous [359]. With this new amendment, the D/V Compartment Hypothesis predicts that 1. A band of wg-on cells should arise at the D/V line via the Fng-N-Dl-Ser (Fringe, Notch, Delta, Serrate) cassette. 2. Wg would diffuse away in both directions.

3. Trapezoids could then orient themselves relative to the slope of the gradient. One aspect of this hypothesis does ring true. Trapezoid polarity is affected by some of the same Wgpathway genes that control the planar polarity of bristles [24, 276, 1449, 3630], wing hairs [1638--1640], and leg joints [1641, 1810, 4716]: arrow (LOF) [4573], fz (LOF or GOF) [3912, 4170, 4365, 4873], and dsh (LOF or GOF) [367, 4278, 4365, 4573, 4873]. Similar changes can be achieved by genes in the branch of the pathway that is normally uncoupled from polarity [2883, 3563], although those alterations may be indirect [4573]: arm (LOF or GOF) [4573] and sgg (GOF) [4365, 4573]. However, Wg is not expressed at the equator [2631, 4390], so another Wnt may be dictating tissue polarity [25, 359, 2781, 4263]. Alternatively, the non-Wnt protein Four jointed (Fj) could be the eye’s D/V polarizer [447, 483] (cf. the wing [4852]). Not only is Fj expressed in the expected gradient proﬁle (Fig. 7.3) but it also seems to integrate inputs from all three pathways that are implicated in equator induction [4173]: Wg [4573], Notch [3258], and JAK-STAT [2618, 4851]. The notion that the eye uses the same Fng-N-Dl-Ser device as the wing was experimentally conﬁrmed by

206

wild-type mutant

a

IMAGINAL DISCS

Iro-C: OFF

mosaic eye

Iro-C

null

equator (ectopic)

ON ON

in v erte d

b

equator

OFF ON

V

NSer X?

c

P

D-type

eye

V

D

Ser

and

Pc-G

P

A

OFF

Iro-C

D

Fng

?

V-type NDl

OFF

ON

A

D

Ser

V

?

Dl

Dl

d Iro-C fng N(act)

D V cells

f 1 1

0

1 0

0

?

1

0 0

0 and

1

X?

0

1

0

(deaf)

D-type

0

0

1 X?

1

(deaf)

0

and 1

1 1

(mute)

1

(mute)

e

0

?

V-type

CHAPTER SEVEN. THE EYE DISC

a spate of articles in 1998 [774, 1080, 3258]. The chief difference is that fng is regulated negatively (Iro-C fng), instead of positively (Ap fng). Link 1:

207

Link 2:

Iro-C fng. Within the eye anlage, fng is mainly expressed ventrally [3258], starting in 1st instar [774]. This expression vanishes when caup [1080] or mirr [774, 4797] is activated widely (GOF test). When Iro-Cnull clones arise in the D area, these cells derepress fng [696] (LOF test; cf. similar effects in mirr LOF discs [4797]). The derepression of this V marker afﬁrms that Iro-C is a D-vs.V selector switch. The downstream status of fng is supported by the fact that mirr must act via fng to induce equators: extra equators form around mirr LOF D clones (which have a fng on/off edge) but not around mirr LOF fng LOF D clones (which do not) [4797]. Mirr’s ability to repress tar-

get genes may be due to recruitment of a chromatin-remodeling co-repressor [941]. fng on/off boundary N-act. Notch is normally activated (N-act form) at the D/V boundary because (1) the Notch target gene mβ is expressed there as early as 2nd instar [1080], and (2) a reporter gene with Su(H) binding sites is likewise activated there [1080]. Ubiquitous expression of fng or caup (via eyeless-Gal4) stiﬂes eye development [774, 1080, 3258]. Eyes can be rescued by jointly expressing N-intra – Notch’s constitutively active intracellular fragment [1080]. The fng on/off boundary is essential for eye development from late-1st to mid-2nd instar [774]. As in the wing, Fng appears to disable N’s reception of Ser signals and to enable reception of Dl signals [3245, 3258] – a bias that limits N-act to the D/V border [1988].

FIGURE 7.4. Control of ommatidial polarity by the Iroquois Complex (Iro-C).

a. Iro-C genes are expressed in the D, but not V, half of the eye ﬁeld during normal development (disc schematic below). The equator (horizontal line) arises at this on/off boundary, and all photoreceptor trapezoids (cf. Fig. 7.2) point away from it. When an Iro-Cnull clone (black) is induced in the D area, it causes wild-type trapezoids within ∼7 rows to invert their polarity. (The eye is ∼33 rows high.) Hence, an ectopic equator emerges at the clone edge. b. Generic eye cell (cf. Fig. 2.7 for icons) showing the main control circuit for emitting an unknown diffusible factor (X) that regulates polarity. Abbreviations: Dl (Delta), Fng (Fringe), N (Notch; “act” = activated form), Pc-G (Polycomb-Group genes), Ser (Serrate). NSer and NDl are hypothetical Fng-modulated forms of Notch that are receptive to Ser or Dl [990, 1250, 1988]. The circuit works as follows. If Pc-G products are sufﬁciently low, then Iro-C genes can be expressed (e) [3079]. In that case, the cell adopts D-type identity and expresses Dl (by an unknown route) but not Ser or Fng. D-type cells can receive Ser signals (from across the D/V line). If Iro-C genes are off (f), then the cell adopts V-type identity and expresses both Ser and Fng. In that case, Fng blocks the Ser-N interaction, so V-type cells can only receive Dl signals. The circuit allows two types of dialog: Ser-signal “speakers” with NSer “listeners” or Dl-signal “speakers” with NDl “listeners”. Both conversations should turn on Factor X. Factor X uses Frizzled as a receptor (cf. Fig. 7.5) [869, 1194, 4366]. c. Eye portion of a mature left eye disc. Shading denotes D (dark; Iro-C = on) vs. V (light; Iro-C = off) compartments. Note that this eye was rotated 90◦ counterclockwise relative to the eye in a (cf. compasses and disc icons) to facilitate comparison with the wing disc’s circuit diagram (Fig. 6.8). d. Enlarged part of the D/V border (box in c). In this single ﬁle, cells on the D side express Iro-C genes, while cells on the V side express fng. Interactions between cells in e and f activate Notch at the D/V boundary. The width of the N(act) stripe (here shown spanning 4 cells) is not known exactly. e, f. Schematics of cells ﬂanking the D/V line. The V cell is drawn as if ﬂipped around to face the D cell. States of variables (cf. b) are recorded as “1” (present and active) or “0” (absent or inactive). Black circles indicate determining factors. “Mute” and “deaf” denote inability to send or receive particular signals (Ser above, Dl below). The functioning of the circuit is explained under b above. Panel a is a schematized adaptation of a photo in [696]. Circuitry in b is compiled from [696] (Iro-C), [3079] (Pc-G), and [774, 869, 1080, 1194, 3258] (Fng-N-Ser-Dl). Panel d incorporates data from the same sources. N.B.: The D/V circuitry of the eye differs from that of the wing insofar as fng is expressed ventrally instead of dorsally (cf. Fig. 6.8). This difference is attributable to the 180◦ rotation that the eye disc undergoes after its inception [774, 4146] (cf. Fig. 7.1a legend). The “Iro-C Ser” link is simpliﬁed from the “Iro-C Fng Ser” route [696, 774, 3258], and the full path is probably even more complex. This model does not explain how equators arise from ectopic furrows that are triggered without altering Iro-C expression [722, 723, 4167, 4648]. Other key players in this scenario may be (1) Lobe (Lobe LOF causes mirr to turn on in the V half and hence suppresses ommatidia [758, 2560]) and (2) teashirt (tsh LOF suppresses ommatidia in the D half) [3973].

208

Link 3:

Link 4:

IMAGINAL DISCS

Fng Ser. Like fng, Ser is expressed in the V half of the eye region [774, 1080, 3258], more intensely at the D/V border as development proceeds. Ser turns off in fng null V clones [774] and turns on in fng GOF D clones when the latter are near the D/V line [3258]. Iro-C Dl ? Dl is expressed in the D half of the eye region, most intensely at the D/V border [774, 1080]. No link with the Iro-C or any other upstream gene(s) has yet been shown.

Upstream of the Iro-C itself, several links are known to operate throughout most of larval life: Link 5:

Link 6:

Wg Iro-C. In early 2nd instar, wg is expressed coincidentally with ara-caup in the D half of the eye rudiment [696]. Later this wg-on zone shrinks to the edge of the D realm, while ara-caup persists throughout it (Fig. 7.3). When the Wg pathway is shut off by dsh LOF clones during either period, expression of ara and caup ceases in the clones [696]. When the pathway is ectopically turned on by sgg LOF clones, ara-caup turns on (except in the V region) [696]. Wg likewise controls mirr: mirr expression disappears from the D half of wg LOF discs and arises ectopically in the V half in wg GOF (dpp-Gal4:UAS-wg) discs [1781]. Hh Iro-C. Like wg, hh is expressed within a progressively shrinking D domain [696]. When the Hh pathway is shut off by smo LOF clones, ara and caup turn off in the center of the clone [696]. When the pathway is constitutively activated by ptc LOF clones that are induced in 2nd instar, mirr turns on inside the clone and in nearby cells, although clones induced at later times have no such effect [696].

The investigations that established the above links also discovered something odd about fng: homozygous fng+ clones affect ommatidial polarity in a heterozygous fng+ /fng null background [1080]. This ﬁnding implies that differences in dosage (2 vs. 1 copy of fng + ) sufﬁce to tilt ommatidia in one direction vs. another [4762]. Ergo, an analog step must exist somewhere in the polarity circuitry. That step was revealed in 1999 [869, 1194]. It also uses Notch, but in an entirely different way from

the equator module [359]. To explain how this gadgetry works, it is ﬁrst necessary to brieﬂy outline the process of ommatidial maturation.

A morphogenetic wave creates the ommatidial lattice During 3rd instar, which lasts 2 days, a wave of cell differentiation sweeps across the eye disc epithelium from posterior to anterior. At its leading edge is a D-V groove called the “morphogenetic furrow” (MF) [3539]. Ahead of the MF are undifferentiated cells and scattered mitoses. Behind it are columns of nascent ommatidia. Columns emerge at a rate of one every ∼1.5 h initially, but the rate accelerates to one per hour when the MF reaches the anterior region [228, 4715]. A mature disc has ∼26 columns, and ∼7 more are added during the ﬁrst 10 h of the pupal period to complete the lattice [602, 1287, 4715]. The discussion here and in the next few sections focuses on the individual ommatidia. The way in which the lattice as a whole arises is considered subsequently. Each incipient ommatidium undergoes a stereotyped series of changes after it leaves the MF (Fig. 7.5b) [186, 4355, 4364, 4712, 4715]. In the stages listed below, all columns are numbered starting at the MF (0) and counting posteriorly, with precursor cells being denoted by a “p” sufﬁx: 1. Rosette (column 0): A ring of 10–15 cells around a core of 4–5 cells appears within the MF. 2. Arc (columns 1 and 2): The rosette’s anterior cells are thought to return to the population of undifferentiated cells, leaving the posterior ones as a distinct arc. At the center of this chain is R8p, which is ﬂanked by R2p and R3p on one side and R5p and R4p on the other, with “mystery” cells at the extremes [4366]. 3. Precluster (columns 3 and 4): The arc zippers shut, so that R3p and R4p touch for the ﬁrst time. Besides R2–5p and R8p, the incipient precluster retains a few mystery cells that merge into the background after R1p and R6p are added. 4. Cluster (column 5): Like R1p and R6p, R7p is recruited from cells that have just ﬁnished dividing in a “mitotic band” at ∼ columns 3 and 4 [185, 625, 629, 1869, 3539, 4355]. At about this time, the clusters start to rotate (clockwise or counterclockwise) [4873]. Consistent with the above sequence, the ﬁrst cell to express neural antigens is R8p, followed by the R2/5p pair, R3/4p pair, R1/6p pair, and ﬁnally R7 [4361, 4364, 4715]. Cone cells are incorporated just after the Cluster Stage,

CHAPTER SEVEN. THE EYE DISC

whereas pigment cells (primary, secondary, tertiary) and bristles are added later (after pupariation) [602, 605, 1287, 1289, 4355, 4715]. Until about the Cluster Stage all nascent ommatidia are bilaterally symmetric [4355], and R3p and R4p are equivalent in all discernable respects. Now these cells diverge: R4p loses its attachment to R8p, but R3p does not [4355, 4364, 4873], and both cells undergo a shift (dorsal in the disc’s D half and ventral in the V half ). Henceforth, each cluster has a handedness (“chirality”) that dictates its direction of rotation [359, 1639]. Rotations occur in mirror symmetry so that D vs. V trapezoids later point in opposite directions (Fig. 7.5) [2883, 4715].

D-V polarity depends on a rivalry between R3 and R4 precursors R3p and R4p are instrumental in steering the entire ommatidium one way vs. the other. This conclusion was reached by analyzing various kinds of frizzled (fz) mosaics. In virtually every case, the R3/4p cell that becomes R3 (the trapezoid tip) is the one with greater Fz activity, regardless of the disposition of genotypes in the other ommatidial cells [4366, 4873]. This rule was also obeyed regardless of absolute Fz levels. Thus, the same outcome was seen when the high-vs.-low discrepancy was created by (1) fz+ vs. fz null , (2) two copies of fz+ driven by a sevenless (sev) enhancer vs. fz null , or (3) two copies of sev-driven fz+ vs. fz+ . Similar studies with Notch and Delta mosaics afﬁrmed the key role of the R3/4p pair. An uncommitted R3/4p cell can be forced to become R3 by suppressing its Notch pathway (via a chimeric Su(H)-Engrailed repressor) [4366]. Conversely, activating the Notch pathway (via a sev-driven N-intra) enforces the R4 state [4366], as does “muzzling” the cell by making it Dl null while leaving its rival alone (Dl+ ) [1194, 4366]. Evidently the two R3/4p cells compete. The one that emits more Dl becomes R3. Epistasis experiments show that the Notch pathway acts downstream of the Fz receptor [869, 1194, 4366]. In fznull ﬂies whose R3/4p cells carry 2 or 3 doses of Notch, the R3/4p cell with 3 doses invariably trumps its 2-dose rival and becomes R4 (48/48 cases) [4366]. Amazingly, this factor of 1.5 sufﬁces to decide the R3-vs.-R4 contest with ∼100% ﬁdelity. Evidently, a small analog imbalance can be ampliﬁed to produce an all-or-none binary output. The ampliﬁcation is thought to involve a positive feedback loop (“N-act” is the activated N receptor): “Dl N-act” between cells and “N-act Dl” within cells (Fig. 7.5g) [869, 1194, 1195, 4366]. The net effect of this loop is that the more one cell signals, the more the other cell

209

is prevented from signaling [1613]. The dialog becomes a monolog, with one cell as the speaker and the other as the listener. A similar “Delta-Notch Flip-Flop” was invoked in the Mutual Inhibition Model of bristle patterning (cf. Fig. 3.6). Serrate is excluded from both circuits [1194, 4366, 4859]. In normal development, the R3/4p cell that is closer to the equator becomes R3. Putting this fact together with the above clues suggests the “Scalar Model” for ommatidial polarity (Fig. 7.5e) [359, 869]. 1. Confrontation of Iro-C on and off cells activates the Notch pathway (via the Fng-N-Dl-Ser module) in a stripe of equatorial cells: Iro-C-on/off N-act. 2. The equatorial cells emit a diffusible molecule (factor X): N-act X. 3. Diffusion of X away from the equator forms a gradient in each half of the eye. 4. Fz senses the X ligand and represses Notch proportionally. Repression could be mediated by Dsh [869, 1194], which acts downstream of Fz [152, 422, 2326]. Dsh can bind and block the Notch receptor [151]: X Fz Dsh N-act. The bias may also be due to a Notch-independent effect on Dl transcription [1194, 1195]: Dsh RhoA JNK dJun Dl [423, 2781, 4170, 4566]. Other agents (of uncertain linkage) include Dachsous [21], Expanded [367], Misshapen [3261], Prickle [1641], Starry night (a.k.a. Flamingo) [704, 4430], and Van Gogh (a.k.a. Strabismus) [25, 4716]. 5. Within each cluster, the two R3/4p cells compete via the trans-cellular circuit “Dl N-act Dl”. Whichever cell expresses more Delta – and activates its Notch pathway less – “wins” and becomes R3. The “loser” becomes R4. Van Gogh is required for a cell to adopt the R4 fate [4716]. 6. Within each pair of R3/4p cells the cell closer to the equator wins due to the bias from Step 4. 7. R3/4 asymmetry dictates each ommatidium’s chirality and direction of rotation (but see [4716]). One obvious question is whether Step 1 interferes with Steps 4 and 5, because they all use the same Notch pathway in different ways. Interference at the equator is apparently avoided by ﬁnishing Steps 1–4 before Steps 5–7 begin: the Notch target gene mβ is on at the equator ahead of the MF, but is diffusely expressed behind it [871, 1080]. The bias introduced in Step 4 relies on the “decoding” logic of a canonical gradient model. That is, the amount of morphogen is measured and “interpreted” as a certain output (Fig. 7.5e). The initially large distance

210

IMAGINAL DISCS

MF MF: Morphogenetic Furrow MB: Mitotic Band

D P

A

34

V

a

c 4 3

7

asymmetry

m4 Arc

4 5 Precluster ...

6

3/4?

equator

f Type-2

N

N

N

Dl

Dl

Dl Dl

D V

3 3

3/4? 3/4?

3 4

Dl Fz Dl

Fz

3/4?

N Dl

N Fz N

Fz

3/4?

Dl

Dl

Vector Model

Bias?

senses level

Dl

Dl

3/4? 3/4?

N

3/4?

4 3

Dl

3/4?

34

N Dl

3/4?

Dl

X?

3/4?

N

3/4?

90 rotation

4

Dl

g

Dl

senses 3/4? level

D Vo

Cluster

Scalar Model

d

3 4

7

Dl Fz N

Type-1 Bias?

8

3/4?

Dsh

asymmetry 1

N

Rosette

e

2 8 5

Delta Dl

b

MB 3 2

Dl

m 3

o

90 rotation

4

CHAPTER SEVEN. THE EYE DISC

between the R3/4p cells (Arc Stage) would allow them to gauge local differences in X levels at a high signalto-noise ratio [3563]. The levels could then be recorded in some more permanent “memory” before the two cells close the gap, touch, and interact (Precluster Stage). Alternatively, Step 4 might use a different sort of bias (Fig. 7.5f: “Type 2” vs. “Type 1”). The “Vector Model” was proposed by Andrew Tomlinson and Gary Struhl [4366], who conducted the Fz-N-Dl experiments. Except for Step 4, it is like the Scalar Model. The new assumption is that differences in X levels can be sensed across the span of a single cell. For example, unoccupied Fz receptors might bind Dl in cis, while X-bound Fz receptors bind N in cis. As a result of this asymmetric “capping” (Fig. 7.5f), the equatorial side of every R3/4p cell would have more N and less Dl than the polar side.

211

When the two such R3/4p cells touch, the one closer to the equator will have more Dl and less N at the interface and hence should always become R3. This model agrees more closely with what is known about planar polarity in general [22], although Notch signaling only seems linked to cell polarity in the eye [4366].

R1--R8 cells arise sequentially, implying a cascade of inductions In their 1976 “neurocrystalline” paper, which disproved Bernard’s Lineage Hypothesis, Ready et al. proposed a model that relied on local cell interactions [3539]. Their “Crystallization Model” is a variation on the notion of sequential induction [91, 2409, 2448, 2716, 3645]. In this particular scheme, the MF acts as a template interface where na¨ıve (anterior) cells “read” their location relative to

FIGURE 7.5. “Symmetry breaking” in nascent ommatidia. Precursor cells are denoted by a “p” sufﬁx in the legend (omitted from

ﬁgure) and equivalent pairs by a slash mark (e.g., “R3/4p”). Abbreviations: Dl (Delta), Fz (Frizzled), N (Notch). a–c. Histological appearance of photoreceptor clusters during development. d–g. Control of D/V chirality by Fz and N signaling. Cubes are individual cells, and inscribed circles are nuclei. Na¨ıve R3/4p (“3/4?”) cells are shaded (left). Committed R3p and R4p cells (far right) are black or white, respectively. See also App. 7. a. Mature left eye disc. Dots are ommatidial sites. Vertical stripes are the morphogenetic furrow (MF) and mitotic band (MB; see [185, 4291, 4293, 4355] for details). Ahead of the MF are scattered mitoses (not shown). Compass (at right) gives axes in the fate map (cf. Fig. 7.1). “Rows” and “columns” run along A-P and D-V axes, respectively. b. Stages in development of a cluster. Because the MF moves as a wave from P to A, this series is manifest in each row (magniﬁed from box in a). Shading indicates future photoreceptors. Numbers denote the prospective fate of precursors (“3” = R3, etc.), not their current state of determination (which may be indeﬁnite). Mystery (“m”) cells leave the arc as it collapses into a precluster [186, 4364, 4715]. The sequence of depicted stages does not correspond exactly to the columns as numbered in the text: some transitional stages are omitted. For example, rotation is more gradual than implied here: it starts after the precluster stage (∼ column 3–6) and ends by the 4-cone stage (∼ column 15) [4361, 4364] (T. Wolff, pers. comm.). c. Clusters become asymmetric at the 2-cone cell stage [4355, 4873] (≈ column 11 [4364]) when R4p severs its connection to R8p and undergoes a slight shift (D or V) jointly with R3p. At about this time, the clusters rotate clockwise or counterclockwise [777, 1639, 2794, 4873] on either side of the equator (thick dashed line). Trapezoid shapes arise later. (Only rhabdomeres really adopt this shape, not whole cells as shown here.) d. Posterior (eye) part of the disc. The polarizing signal (X = E-shaped ligands) is assumed to be under N control (cf. Fig. 7.4) and to form 2 gradients by diffusing from the equator. Fz is the receptor for X. At the Arc Stage, the na¨ıve R3/4p cell nearer the equator should sense a higher level of X (dotted line) and hence activate more of its Fz receptors [4366, 4873]. e. According to the Scalar Model [869], this higher Fz activity represses N (via Dsh) more strongly than in the rival cell. When the cells touch (Precluster Stage, d), this bias causes the cell nearer the equator to have less active N, so it wins the “shouting” contest (i.e., whoever has the most Dl) to become R3. f. The Vector Model [4366] invokes a different kind of bias (Type 2 vs. 1). The side of the cell that faces the equator should have more of its Fz receptors occupied than the other side. If X-bound Fz receptors recruit N while vacant Fz receptors recruit Dl, then each R3/4p cell will have more N on its equatorial side and more Dl on its polar side. When the two such R3/4p cells meet (d), the one nearer the equator will have more Dl at the interface and so will always win and go on to become R3. (Other variations on this scheme are possible.) g. Enlargement of R3/4p pairs after these na¨ıve (gray) cells abut (Precluster Stage). The outcome of the contest (black “3” or white “4”) is preordained by the bias introduced earlier (e or f), although no bias is depicted here (g). The positive feedback loop between the cells constitutes a “Delta-Notch Flip-Flop” device, whose instability eventually forces the cells to adopt binary (alternative) R3 vs. R4 fates (Cluster Stage at right) by amplifying the small initial bias [2883]. Relative locations of MF and MB in a are based on [604, 3539]. Panels b and c are adapted from [293, 604, 3539, 3563, 4355, 4715, 4873], although not all the depicted stages (b) occupy successive columns [605, 2887], and the mitotic band may actually span several columns [185, 228]. Models and schematics in d–g are modiﬁed from [359, 447, 869, 4173, 4366]. For details of rosette geometries, see [184, 4712]. For critiques of earlier models, see [3562, 3563]. See also App. 7.

212

already committed (posterior) cells and adopt fates accordingly. Cell differentiation in the eye appears to be unrelated to cell lineage. The final cell type may thus be determined according to the lattice position into which the cell is recruited. This is reminiscent of the growth of a crystal, in which the leading edge of the pattern serves as a template on which new elements are incorporated. Rather than being required to assess its position independently, a cell joining the ensemble could use the information available at the growing edge to determine its role. This assessment may be based on combinatorial cell contacts, the information being mediated by surface molecules, as in the ‘‘antigen-antibody’’ model of Tyler [4412] and Weiss [4590]. According to such a scheme, an undetermined cell ahead of the furrow might display a set of antigens. ‘‘Antibodies’’ on cells at the leading edge would bind a specific subset of these antigens, thus informing the newly added cell of its role and causing it to display an appropriate set of antigens in turn, propagating the pattern. [3539]

According to this model, the crystallization process would be initiated at the back of the eye by a special group of cells, and the remaining tissue should be unable to form ommatidia properly if this “seed crystal” is removed. In the 1960s, Richard White used this logic to prove the existence of such a nucleation center in the mosquito eye [4628, 4629]. He explanted prospective eye tissue with or without the “optic placode” (a thickening at the rear of the eye ﬁeld) and found that ommatidia develop only when the placode is included in the explant. Hence, the placode behaves like a seed crystal. White was also able to prevent differentiation ahead of the MF in situ by implanting a strip of foreign epidermis in its path. In one case where the implant failed to reach the edge of the host’s eye ﬁeld, the MF traveled backward beyond the barrier, thus revealing an ability to move in an A-to-P as well as a P-to-A direction. This ability is also consistent with the Crystallization Model because the MF should be free to travel in any direction after being deﬂected (like a ripple in a pond [2036]). When similar experiments were performed in Drosophila, different results were obtained. In 1986, Lebovitz and Ready reported that anterior pieces of eye discs (ahead of the MF) can make ommatidia when explanted [2452]. To rule out the possibility that the ectopic MF arises as a consequence of wounding, they cut the disc diagonally and showed that ommatidia tend to differentiate in a wave from P to A, rather than in a wave that is parallel to the wound edge. Their conclusion that wounding is irrelevant cannot be considered deﬁnitive, however, because hedgehog expression was not monitored. As discussed in Chapter 5, contact with

IMAGINAL DISCS

the peripodial membrane might be triggering patterning events de novo at particular sites [773, 1472]. The fact that killed the Crystallization Model was not the ability to incite ectopic MFs, but rather the wider-than-normal spacing of ommatidia in Egfr GOF (“Ellipse”) eyes [179--181, 2355, 2493, 3410] (cf. ato LOF [4621] and rap LOF [2145]). These lattice gaps are hard to reconcile with any template-based process [1048] because the ability of clusters to develop far from one another (i.e., in isolation) argues that ommatidia must be self-assembling units [185, 4360]. In 1986, Ready, Tomlinson, and Lebovitz [3540] salvaged the main premise of the Crystallization Model (viz., that na¨ıve cells adopt fates based on the committed cells they happen to touch). Even if the entire MF does not act as a template, a template-like process might still be occurring within the conﬁnes of each nascent ommatidium. The “Combinatorial Cascade Model” that they devised focuses on intra-ommatidial signaling, while ignoring the issue of interommatidial patterning (Fig. 7.6e, f; see [3645, 4412, 4590] for earlier models of this kind): The crystal-like regularity of the Drosophila compound eye thus does not arise like a crystal. . . . The orderly sequence of ommatidial assembly, together with the lack of cell migration revealed in the genetic marking experiments, suggest that the fates of undetermined cells in the retinal epithelium are determined in place by their interactions with neighboring cells that have already been incorporated into a precursor. At each stage of assembly, the stereotyped morphology of the precursor presents defined niches to surrounding epithelial cells which could supply the cues necessary for a cell to determine its appropriate fate.

Tomlinson and Ready expounded on this theme in 1987 [4364]: The ommatidium appears to be an autonomously assembling unit. The stereotopy of the assembly process and the precise cellular contacts made, coupled with the autonomous assembly, point to local cues for pathway selection. . . . Taken together, the patterns of ommatidial development suggest that undetermined cells are directed into specific pathways by ‘‘reading’’ the identities of the cells they contact. A particular combination of cell types may specify a particular fate. For example, a cell in contact with R1, R6, and R8, would have an unambiguous cue to become R7. Once a cell has determined its fate, it must then display its identity so the next cells in line can make informed choices. A simple logic of cell contacts may direct pathway choice in the developing ommatidium.

In 1989, Cagan and Ready added cone cells and pigment cells (PPC, SPC, TPC) to this scheme based on geometric clues [602]: (1) a cell that contacts a cone cell becomes a

CHAPTER SEVEN. THE EYE DISC

213

PPC, (2) a cell that contacts PPCs from two ommatidia becomes a SPC, and (3) a cell that contacts three PPCs becomes a TPC.

signal is probably not Rough itself because Rough has a homeodomain.

But the final cell (R7) is induced by the first one (R8) Order emerges in the eye as specific and stereotyped contacts are made between retinal cells. . . . The fact that these contacts are consistently seen in the early development of each pigment cell suggests that they are important for cell determination. . . . Each cell’s position appears to direct its pattern of gene expression, which in turn directs its fate. Ommatidial development progresses by evolving new determinative positions that allow for new cell fates. In this manner, positional information, encoded in stereotyped cell contacts, directs the patterns of gene expression which drive the selfassembly of the eye. [602]

As mentioned above, neural antigens are expressed by photoreceptor precursors in a deﬁnite order (Fig. 7.6b) [4361, 4364, 4715]. In the series below, “ ” refers to time. Thus, R8 manifests neural characteristics ﬁrst and R7 manifests them last. Neural features:

R8p R2/5p R7.

R3/4p

R1/6p

A priori, it was natural to think that this sequence might reﬂect a causal chain of events [4769]. In the Combinatorial Cascade Model, each step (except the ﬁrst) was thought to involve several signals, with different signals (and receptors) being used at successive steps because they come from different cell types. In each of the steps below, “ ” signiﬁes “induces”. Step 1 (direct): Step 2 (Boolean): Step 3 (Boolean): Step 4 (Boolean):

R2/5p. R8p {R8p and R2/5p} R3/4p. {Some combination of R8p, R2/5p, and R3/4p} R1/6p. {Some combination of R8p, R2/5p, R3/4p, and R1/6p} R7p.

Thus, two general kinds of mutations were expected: (1) mutations that block emission of signals, and (2) mutations that block receipt or transduction of signals. Such a model predicts two classes of mutation, one class which interferes with the normal expression of cell-type identity signals and a second which prevents cells from properly reading these signals. [4363]

The gene rough ﬁt neatly into this scheme [3670, 4356, 4358]. The only cells that need rough are the R2/5p pair. When they are rough LOF , the R3/4p and R1/6p cells fail to mature as neurons [1783, 4361]. Evidently, R2/5p cells recruit neighbors into the cluster by emitting a signal. The

Curiously, R7s were found to arise normally in rough LOF eyes [1783]. The implication was that R7 is not downstream of the rough-dependent signal but rather relies on a signal that comes from R8 alone. (Note that rough LOF R3/4p and R1/6p cells could still inﬂuence R7p despite their immaturity, and indeed they do, as discussed below [4367].) The model can easily be revised to incorporate two separate signals from R8p: Step 1 (direct): Step 2 (Boolean): Step 3 (Boolean): Step 4 (direct):

R8p (early signal) R2/5p. {R8p and R2/5p} R3/4p. {Some combination of R8p, R2/5p, and R3/4p} R1/6p. R8p (late signal) R7p.

The “R8p R7p” induction does in fact occur, and its components manifest the expected speciﬁcity [4360]. Indeed, the ligand and receptor for this tˆete-`a-tˆete are used nowhere else in the ﬂy. The ligand is a 7-pass transmembrane protein encoded by boss (bride of sevenless) [1732], and the receptor is a tyrosine kinase encoded by sev (sevenless) [428, 1677]. As usual for RTK receptors (cf. Fig. 6.12), Sev is activated by Boss (vs. inhibited) [1733, 2319, 2450, 3671, 4359]. LOF mutations in either gene eliminate R7 [4890], but no other LOF effects are seen elsewhere [604]. Boss is peculiar insofar as it is a 7-span membrane protein that is totally engulfed by the receiving cell [601, 2318, 4890], and Sev is odd insofar as it is cleaved and rejoined to form an external loop that is anchored at both ends to the membrane [227, 2318, 2991, 3342]. Otherwise, these partners seem to work like an ordinary lockand-key device (cf. Fig. 6.12), albeit customized for one cellular chat. Closer inspection, however, reveals nuances. Genetic mosaics show that boss is only needed in R8 [3569] and that sev is only needed in R7 [632, 1726, 4363]. This result is not surprising for boss because R8 is the only boss-on cell in the cluster [2319, 4459], but it is puzzling for sev because sev is on in other cells that touch R8 [2450] – namely, R3/4p and weakly in R1/6p (but not at all in R2/5p) [194, 227, 230, 4360].

Various restraints prevent more than one cell from becoming R7 Why don’t the other Sev-expressing cells that touch the Boss-expressing cell become R7s? Their unresponsiveness is attributable to the transcription factors Rough

214

IMAGINAL DISCS

D P

A

a

b

MF

neural dJun Boss Sev Sev*

V

0 hours

7.5

y ... z ... z ... 7 ... C ...

x ... 8 ... 8 & x ... y ... 8 ... y ...

0 columns

15

8 8 8 8 8

taken up by R7

5

10

Argos Spitz

T! R A ST

P C

7

STOP!

y x 8 y x

z y x 8 y x z

STOP!

STOP!

y

z

x

8 8

STOP!

x x 8 x x

x y z

z Spitz Argos

THEN BECOME:

7

f z

!

IF YOU ARE TOUCHING:

STOP!

7

8

8

e

R2/5p R3/4p R1/6p Cone

y

Combinatorial Cascade Model

? 8 ?

x: y: z: C:

x

d

Stop the Clock! Model

15

z y x 8 7 y x z

STOP

c

STOP!

7 ...

7 ... C ... 7

Key:

MF

22.5

and

READ!

READ!

READ!

READ!

?x 8 x ?

? y x 8 y x ?

z y x 8 ? y x z

z y x 8 7 y x z

8 ONLY

x ONLY

8&x

8&z

x

y

z

7

7

CHAPTER SEVEN. THE EYE DISC

(in R2/3/4/5p) and Seven-up (Svp, in R1/3/4/6p) – neither of which is expressed in R7p [4890]. Thus, all outer R cells (R1–R6) can become R7s in doubly mutant svp LOF rough LOF clones [1783]. Cone cells normally express Sev but do not touch R8p [230, 4360] or turn on svp [2888]. They can be induced to adopt an R7 fate by ubiquitously expressing boss (R1–R6 persist intact) [4459] or by artiﬁcially activating their Sev pathway [225, 229, 1049]. Hence, their failure to become R7s in wild-type ﬂies is solely a consequence of their physical separation from R8p. Overall, therefore, the data suggest that a cell must possess four traits to become an R7 [600]. Each trait is exhibited by a certain set of cells (in braces below: Cp = cone precursor, M = mystery cell) [897, 938, 4459].

215

Must have Sev:

Set 1:

Must lack Rough:

Set 2:

Must lack Svp: Must touch R8p:

Set 3: Set 4:

{R1p, R3p, R4p, R6p, R7p, Cp, M}. {R1p, R6p, R7p, R8p, Cp, M}. {R2p, R5p, R7p, Cp, M}. {R1p, R2p, R3p, R4p, R5p, R6p, R7p}.

The overlap between Sets 1, 2, and 3 deﬁnes the “R7 equivalence group” {R7p, Cp, M}, whose members are competent to react to Boss [1049, 1678]. Within that pool, only a single cell satisﬁes the ﬁnal criterion (Set 4) and hence becomes R7 [3540]. Additional prerequisites exist. For example, receipt of a Delta signal from R1/6p turns out to be essential for

FIGURE 7.6. Models for recruitment of photoreceptor cells into nascent ommatidia.

a. Mature left eye disc. Vertical stripe is the morphogenetic furrow (MF), which moves from P to A. Compass gives axes in the fate map (cf. Fig. 7.1). In c and e, an imaginary slice of the columnar epithelium (boxed in a) is magniﬁed to show how each ommatidium acquires cells after arising as an arc in the MF. Stages are accentuated by stripes (black = MF; gray ≈ later columns) that correspond roughly to columns 0–4 in terms of cluster morphology, although the patterning cues (Stop! or Read!) may occur later. Cells are drawn as ovals (top view; black = determined; gray = uncommitted) or as narrow rectangles (side view). b. Temporal aspects of photoreceptor development. Abscissa is calibrated in hours (25◦ C) and in columns of ommatidial clusters relative to the MF (origin). Bars followed by “...” signify onset (bar) and persistence (...), whereas bars alone indicate total duration. “Neural” means neuron-speciﬁc epitopes recognized by 22C10 and anti-HRP antibodies [1948] (but see [228] for a different pacing and [2322, 4712] for a different order); “dJun” is expression of dJun (duration ≈ 4 h, except cone “C” cells where it lasts longer); “Boss” and “Sev” are expression of these proteins; and “Sev*” is the period when Sev must be continuously activated for a prospective R7 cell to adopt the R7 fate [2991]. This period (columns 7–10) coincides roughly with the time when Boss is internalized (“taken up”) by R7 [2319]. The mitotic band (not shown; cf. Fig. 7.9) roughly spans columns 3–5 [185, 228, 4712]. Omitted: Delta (timecourse ≈ neural) [184, 3273] (cf. Notch [603]). The R8p cell ﬁrst becomes identiﬁable in column 0 when it expresses Scabrous and Atonal (not shown) [184, 2042]. Atonal is required for R8 differentiation [4621]. The “Irregular chiasm C-roughest” protein (not shown) is expressed by cells in the same order as they are thought to be induced (c) [3570]. c, d. The Stop the Clock! Model [1290, 3557] uses two variables: (1) the “state” of a cell and (2) a “Stop!” signal. Cells are supposed to automatically progress through a series of transcription factors (states), represented here as a clockface whose numbers and letters reﬂect cell fate: 7 and 8 refer to R7 and R8; x, y, and z to outer R cells (cf. key for b); and C and P to cone or pigment cells (c). All cells in each column change state synchronously at ∼1.5-h intervals (gray bands), starting in the MF (Start!) with the R8 state, then x, y, z, R7, and ﬁnally (not shown) C and P. The “Stop!” signal forces cells to keep their current state. It is conveyed by Spitz (white arrows) – a ligand for Egfr. Each cell emits Spitz within ∼1.5 h of receiving a Spitz signal, thus propagating the signal by “sequential induction” (although the exact “talking” order is unknown). R7p breaks the chain because (1) it is told to stop by the earliest cell in the series (R8p) and (2) it also requires a Boss signal (transduced by Sev). Because Spitz is diffusible, its concentration should dwindle at increasing distance from its source. This inferred gradient is depicted to the right of the “x” column (ﬁlled triangle), along with a gradient of the ligand Argos (unﬁlled triangle) – a competitive inhibitor. Spitz and Argos are both produced by whichever cell is signaling at any given time (e.g., R8p in d), but the Argos gradient is shallower because Argos diffuses faster [1288, 1290, 1294, 2331]. Thus, Spitz will be louder than Argos for cells next to the vocal cell (hence stopping their clock) but softer than Argos for cells farther away (hence letting them tick). See [2762, 2811, 3644--3646] for older models in this genre. e, f. The Combinatorial Cascade Model [602, 3540, 4364] uses Boolean combinations of cell-surface ligands to elicit certain states in adjacent cells. The rules are given below. For example, the rule depicted in f is: “if you are touching an 8 cell AND a z cell, then become a 7 cell.” Each signaling cell is thought to exhibit a unique ligand (round vs. square lollipops on 8 vs. z), and each receiving cell is thought to have various receptors. In both models, previously determined cells (black) talk to uncommitted cells (gray and “?”). Both models use “inductive signals,” but the former uses them permissively while the latter uses them instructively [196]. Data in b are from [383, 2991, 4360, 4364, 4715]. Cross-sections in c and e are schematics simpliﬁed from [3540]. See [605, 4890] for a 3-dimensional perspective. The cartoon in f is adapted from [1290, 2351, 3538]. See also App. 7.

216

R7 identity (see below) [4367]. Moreover, members of the R7 equivalence group can adopt the R7 fate only during a deﬁnite time window [604] , whose molecular basis is unknown.

The information content of the inductive signals may be only 1 bit What happens to the would-be R7 cell when it is prevented (by sev LOF or boss LOF ) from becoming R7? Oddly, it becomes a cone cell [1726, 2887, 4362]. (The total number of cone cells remains 4 because the transformed cell displaces a would-be cone cell [4363].) This non-neural default was surprising because a priori one might have expected R7p to start from – and revert to – a neural ground state [27, 1818, 4551]. The deeper question here is: what is Boss’s information content [224, 1049, 3671]? Based on R7’s default state, Boss might just be conveying the message, “Become neural!,” with other factors (such as Notch [4367]) biasing the listener to an R7 identity [3671, 4551, 4790]. Curiously, circumstances were found where a cell can lack Sev and still become an R7. If the complement of R cells is represented as an ordered triplet (a, b, c) where the variables are the numbers of outer (R1–R6)type photoreceptors (a), R7-like cells (b), and R8 cells (c), then a wild-type ommatidium would be “(a = 6, b = 1, c = 1).” A typical svp null ommatidium is “(3, 5, 1),” where the R3/4 and R1/6 pairs have become R7s, and there is an extra outer-type R cell of unknown origin. When svp null clones are induced in sev null eyes, the doubly mutant ommatidia are commonly “(3, 2, 1)” [2888], where R7 and two of the four R7 transformants (R3/4?) are missing, but the other two R7-like cells (R1/6?) manage to differentiate without Sev. Evidently, some other receptor aside from Sev can induce an R7 state. This conclusion was bolstered by the ability of ectopic Svp to transform cone cells (which have no access to Boss) to R7s [1856]. In 1995, the latter transformation was found to depend on Ras-MAPK pathway elements that are shared downstream of Sev and Egfr (cf. Fig. 6.12) [264]. Thus, Egfr was implicated as the accomplice in the svp-mediated conversions. The wider inference was that Egfr and Sev could substitute for one another elsewhere, as long as they exhibit the appropriate onset, duration, rate, and amplitude of signaling. The Combinatorial Cascade Model remained popular throughout this period [229, 605, 1048, 2961, 4714], despite a failure to ﬁnd any receptors besides Sev that are dedicated to deﬁnite R-cell subsets [1288, 4890].

IMAGINAL DISCS

In 1996, Egfr and Sev were shown to be entirely interchangeable [1289], and this ﬂexibility begat a new model [1290, 2243]. The experiments were performed by Matthew Freeman in Cambridge, England. Egfr is naturally expressed throughout the developing eye epithelium (intensely in the MF) [2493, 4843]. Freeman sabotaged the normal Egfr by expressing a dominant-negative Egfr decoy (EgfrDN ) that has the ligand-binding and dimerization domains but lacks the cytoplasmic kinase domain, and he used a hs-Gal4 driver (with UAS-Egfr DN ) to shut off the host Egfr at desired times. He found that virtually any R cell, except R8 [1083, 4799], can be eliminated by disabling its Egfr around the time of the R cell’s inception [1289]. Surprisingly, the inactivation of Egfr also suppressed R7. R7 had been thought to only need a Sev-mediated signal. Apparently, R7 needs two signals – a “primer” via Egfr and a later “boost” via Sev [4322]. Actually, what a cell needs to become an R7 is a high stimulation of its Ras-MAPK pathway. The route that activates the pathway is moot. Thus, Freeman was able to rescue R7 cells in sev null eyes by overexpressing a constitutively active EgfrGOF construct (TorD -Egfr) that bypasses Sev entirely [1289]. Likewise, excess R7s (caused by dMAPK GOF ) can be suppressed by inactivating Egfr (via argos GOF ) [3771]. Another surprise was that later heat shocks stiﬂe non-neural cells. The sensitive periods obey the same sequence as the appearance of these cell types in normal development: cone cells, PPCs, SPCs, and TPCs. Evidently, Egfr mediates the induction of both neural and non-neural cells. This deduction was afﬁrmed by GOF studies: excess R cells, cone cells, or pigment cells can be elicited by expressing TorD -Egfr at successively later stages, and similar effects are seen with spitz GOF [1289, 2493] (but not vein GOF [2493]). Freeman conjectured that Spitz is the only signal that is normally used to recruit all cell types into the ommatidium, except for Boss, which is needed for R7 but can be bypassed. (Delta was not then known to be instrumental [4367].) In each of the following steps, “ ” indicates that the foregoing cell uses Spitz to induce a na¨ıve cell(s) to adopt the next state. For simplicity, the hypothetical series of inductions is written in a linear format to conform to the temporal sequence of inception in normal development. Step 1: Step 2: Step 3:

R8p R2/5p. R2/5p R3/4p. R3/4p R1/6p.

CHAPTER SEVEN. THE EYE DISC

Step 4: Step 5: Step 6: Step 7: Step 8:

R8p (late signal) R7p. All R cells (?) Cone cells. Cone cells PPCs. PPCs SPCs. SPCs TPCs.

This proposal differs from the Combinatorial Cascade Model insofar as a single inducer here acts as a 1-bit “STOP!” or “GO!” command. There are not supposed to be multiple signals acting in combination. Indeed, Freeman boldly entitled his 1996 paper, “Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye.” The dependence of PPCs on cone cells (Step 6) has been proven directly by laser ablation [2859]. Here again, we encounter the Ras Speciﬁcity Riddle (cf. Ch. 6) [3638, 3783, 3942, 4245]: how can the same pathway achieve different outcomes? A plausible answer was put forward in 1994 by Thomas Reh and Ross Cagan [3557], and Freeman built on their premise to craft what may be called the “Stop the Clock! Model” (Fig. 7.6c, d) [1290]. The basic idea, as stated below in excerpts, is that cells progress synchronously through a series of states [1290]: A, B, C, etc. When a cell gets a Spitz signal, it keeps the state that it has at the time, and this state dictates its fate: A = R2/5, B = R3/4, C = R1/6, etc. The states thus furnish a context wherein 1-bit inputs can yield diverse outputs [224, 3287] (cf. Turing machines [872, 1034, 1897, 4409] and L-systems [2557--2559]). The data suggest a model whereby external cues decide the developmental moment a cell will respond to its own evolving internal information. Each uncommitted cell appears to have an internal clock that changes its developmental potency with increasing developmental age. Perhaps this clock represents a progressive cascade of transcription factors. As the complement of factors change, the potency of the cell changes. [3557] Upon activation by [Egfr], a cell would start to differentiate towards the fate appropriate to its developmental stage, indicating that it is the age or developmental history of a cell that is responsible for determining its ultimate fate. This implies that a cell passes through a series of ‘‘states,’’ each representing a potential fate. Each cell state presumably derives from the subset of transcription factors that are present and which can be activated by the Ras pathway. [1290]

A ratcheting device of this kind has been found in neuroblasts. When isolated in a culture dish, embryonic neuroblasts autonomously express transcription factors in a ﬁxed sequence, and their descendants stay in one of the 4 phases [485]: Hunchback (homeodomain),

217

Pdm (POU-homeodomain), Castor (zinc ﬁnger), or Grainyhead (bHLH). The “Stop!” signal in that case must linked to mitosis. The classic example of a transcription factor hierarchy in ﬂies is the “pufﬁng” cascade [121, 123, 246, 2097, 3706], where successive batteries of genes trigger one another in response to ecdysone at metamorphosis [479, 1254, 3059, 4131, 4311, 4422]. Surprisingly, the pufﬁng cascade may be more than just a metaphor here. It is directly harnessed to the MF: the proteins of the Broad-Complex (ﬁrst tier of puffs) are required for ommatidial assembly behind the MF [457, 458], and Ultraspiracle (the ecdysone co-receptor) is needed for proper R-cell differentiation [3198, 4855]. Moreover, Ultraspiracle is inhibited by Seven-up [4856] – a fate-determining factor that also belongs to the steroid receptor superfamily [264, 1856, 2322, 2888]. These correlations suggest that the pufﬁng cascade is literally furnishing the “clock” that Spitz supposedly stops in order to assign cell fates. However, other transcription factors that have no obvious endocrine associations are also candidates for this role and must be considered (see below). Spitz is only active as a diffusible (vs. membranebound) ligand [3829]. It could easily go from R8p to R2/5p to R3/4p if it were to diffuse during the Arc Stage when these cells are in a single ﬁle (vs. later when they are clustered) [196]. Freeman argued that a diffusible inhibitor is limiting the range of Spitz’s effects. If the inhibitor diffuses faster than the inducer, then cells far away will be “immunized” against stray inducer signals. This same sort of Activator-Inhibitor Model was discussed for wing veins (cf. Ch. 6) [3207]. In both cases, the supposed inhibitor is Argos. The following facts support the Stop the Clock! Model: 1. Egfr is required for the differentiation of all R cells except R8 [204, 1083, 1289, 2355, 4781]. R8 is also unique insofar as it requires the proneural gene atonal [2041, 2042], which enables it to found the R-cell cluster [4621]. In Egfr null clones, the PNC spot of atonal expression shrinks to the R8p cell as it normally does, but this reﬁnement is delayed [2493], apparently due to an “Egfr Notch atonal ” chain of control. Nevertheless, R8 does need Egfr later because it cannot retain its neural state without Egfr [1083, 2355]. 2. Each R cell begins expressing Spitz when it becomes neural [4322], and a similar order is seen for Star [1782, 2288] and Rhomboid [1293, 4035]. Star and Rhomboid enable Spitz to be cleaved and released [3385]. For a normal facet to develop, Spitz is absolutely required in R8p, less so in R2/5p and R3/4p, and not at all in R1/6p

218

3.

4.

5.

6.

7.

8.

or R7p [1288, 4321] (cf. consistent data for Star [1785] and Rhomboid [1293]). Evidently, what matters is the local amount of Spitz: fewer than 8 R cells can make enough Spitz for facet completion (albeit perhaps delayed). N.B.: Some of the weak LOF effects might be attributable to Spitz’s cousin Gritz [204, 2108, 4558], which could be acting redundantly here. The ﬁrst step (R8p R2/5p) can be bypassed in null atonal eyes by expressing an activated Egfr ahead of the MF [1083]. Under these conditions, ectopic R1– R7 cells arise but no R8 cells. Likewise, virtually all cells posterior to the MF can be converted to R1–R7 identity in wild-type eyes by expressing spitz there, but no excess R8s are seen [2493]. When R8p is spitz null , it can still muster 4 other cells to form a “precluster,” but it is the only cell therein that becomes neural [1083, 4322]. When R8p is Egfr null , it can still induce neighbors to become neural (presumably by emitting Spitz), but only if they are wild-type (Egfr+ ) [1083]. Like Spitz, Argos is expressed by each recruited R cell as it differentiates [1294, 2331, 3183]. (Argos is also detected in cone cells and PPCs, but not SPCs, TPCs, or bristles [1294].) Both spitz and argos may be targets of the EGFR pathway in the eye, with “Egfr spitz” operating at a lower threshold than “Egfr argos” [2493]. As expected, argos GOF suppresses R cells (all types), cone cells, and pigment cells [515, 1287, 3772, 4233], while argos LOF elicits extra R cells (R1–R6 only), cone cells, and pigment cells [1294, 2331, 3183, 3771, 3772]. The extra cells are recruited from the pool of uncommitted cells around nascent ommatidia [1294] – a conscription that rescues them from the doom of death [515]. Spitz and Argos do indeed have different effective ranges in the eye [1290]. Wild-type cells rescue argos null clones over ∼10–12 cell diameters [1294, 2331], but they rescue spitz null clones over ∼3–4 cell diameters at most [1288]. When an antibody to phosphorylated dMAPK is used to monitor the EGFR pathway, 1–3 central cells per cluster ﬁrst show activity. This activity fades in older columns as staining emerges in the periphery of each cluster [1337]. Expanding rings of this sort are predicted by the model. Timing is clearly important in assigning fates because cone cells can be converted to R7-type cells by activating their Ras-MAPK pathway prematurely [225, 516, 1049, 1273, 1399, 3633]. R7p continues to express an R7-speciﬁc marker gene (klingon) even after it transforms into a non-neural cone cell in sev LOF ﬂies [585, 2887]. Evidently, R7p has

IMAGINAL DISCS

indelible qualities that precede receipt of the Boss signal. Early biases of this sort may constrain R7p to its correct fate [265, 1290]. 9. Ubiquitously activated Notch is unable to summon the expression of speciﬁc E(spl)-C genes in certain tissues at certain times [871]. The selective inaccessibility of these genes to the Notch pathway offers support, albeit indirect, for the idea that speciﬁc genes might become available to the Ras-MAPK pathway at different times. Some properties of the non-neural cells are difﬁcult to reconcile with the above scheme. Cone cells do not seem to need their RTK pathway because they differentiate normally when the transcriptional repressor Ttk88 is expressed throughout the eye epithelium [670]. (See [4367] for an exegesis.) Also unclear is how cone and pigment cells can remain non-neural after receiving a Spitz signal because this signal should lead to the degradation of Ttk88 and hence to neural development [670].

No transcription factor ‘‘code’’ has yet been found for R cells The Stop the Clock! Model envisions a succession of state-speciﬁc transcription factors. Disabling any of them should transform one type of ommatidial cell to another. In other words, there should exist genes that, when mutated, can switch cell fates within the ommatidium, and these genes should encode DNA-binding proteins. Many DNA-binding proteins have indeed been found to play roles in eye development (Table 7.1), but genetic screens have uncovered only two bona ﬁde “homeotic” genes (aside from the effectors of the BossSev pathway): rough and svp [4551]. When expressed ectopically, either Rough [232, 1676, 2223] or Svp [264, 1856, 2322] can cause R7p to adopt an outer-type (R1–R6) identity. If either of these transcription factors can enforce the outer-type state [4790], then both of them would have to be absent for a cell to become R7. This prediction has been tested. Rough and Svp are normally expressed in R2/3/4/5p, and R1/3/4/6p, respectively (Table 7.1). In svp null eyes, R1/3/4/6p cells become R7s. This result is surprising because Rough should still be present in R3/4p, although mosaics reveal that Rough is not needed (functional?) there [4361]. In rough null eyes, R2/5p resemble R1/3/4/6-type cells instead of R7s. The reason appears to be a “Rough svp” link (Link 9 below), so Svp is derepressed in rough null R2/5p cells. If this argument is correct, then rough null svp null clones should convert all outer cells to R7s (via contact with

CHAPTER SEVEN. THE EYE DISC

R8p). The double-mutant phenotype agrees with this expectation [1783]. How are rough and svp turned on or off in R-cell subsets during normal development? We know only part of the story: rough is kept off in R1/6 by BarH1, and svp is kept off in R7p by lozenge. These links (Links 8 and 4) and others are listed below. Each regulatory agent (on the left side of the control sign) is a transcription factor (see [1021, 1293] for other targets). Abbreviations include: lz (lozenge), phyl (phyllopod), pros (prospero), sca (scabrous), sina (seven in absentia), and svp (seven-up). Link 1:

Link 2:

Link 3:

Link 4:

Link 5:

Link 6:

Lz Bar-C. Ectopic expression of lz can activate the Bar-C [938] (= the BarH1 and BarH2 paralogs [1842]), whereas Bar-C gene expression in R1/6p cells is sharply reduced in lz null eyes [897, 938]. Based on its effects here and elsewhere, lz is considered a prepattern gene [1255, 1587, 1653]. Lz dPax2. In lz LOF eyes, dPax2 turns off in cone cells [1256]. The regulation of dPax2 is direct: Lz binds 3 sites in the cone-cell enhancer of dPax2 [1256]. For this 361 b.p. enhancer to turn dPax2 on, it must also bind the transcription factors PointedP2 (effector for EGFR) and Su(H) (effector for Notch) [1256]. Hence, this enhancer uses combinatorial logic: “if Lz and PointedP2 and Su(H) are bound, then turn on dPax2.” How this logic is implemented sterically is not known. Lz pros. Ectopic expression of lz can activate pros [4775], whereas pros stops being expressed in R7p and cone cells in lz LOF eyes [670, 4775]. The regulation of pros is direct: Lz binds 2 sites in the eye-speciﬁc enhancer of pros [670, 4775]. For this 1150 b.p. enhancer to turn pros on, it must also bind PointedP2 [4775]. Lz svp. In lz LOF eyes, svp turns on in R7 and cone cells [897, 938], while in lz GOF eyes, svp turns off in R3/4p [938]. This link must somehow be invalidated in R1/6p cells because they normally express both lz and svp [1255]. Svp BarH1. In svpLOF clones, the cells that normally would express BarH1 (R1/6p) fail to do so [1856]. BarH1 svp. (Converse of Link 5.) When BarH1-off (cone) cells are forced to express BarH1, they turn on svp [1768].

219

Link 7:

Link 8:

Link 9:

Link 10: Link 11: Link 12:

Link 13:

Link 14:

Link 15:

Link 16:

Link 17:

Link 18:

BarH2. Ectopic expression of BarH1 BarH1 (in cone cells) autonomously activates BarH2 expression [1768]. BarH1 rough. When rough-on (R3/4p) cells are forced to express BarH1, they turn off rough [1768]. Rough svp. In rough null mutants, there are more svp-on cells in the ommatidium [1783], apparently because R2/5p cells (svpoff) transform to resemble R1/3/4/6 (svpon). This link appears to be inactivated in R3/4p because those cells express both rough and svp. Rough BarH1. Expression of BarH1 vanishes in rough LOF eyes [1842]. Glass BarH1. Expression of BarH1 vanishes in glass LOF eyes [1842]. Glass sca. In glass LOF eyes, sca is expressed at a subnormal level (although it stays on longer) [4391]. Glass svp. In glass LOF eyes, svp fails to be expressed in its normal complement of photoreceptors [4391]. Glass boss. In glass LOF eyes, boss expression virtually disappears [4391], although sev expression persists [2963]. Glass glass. In glass LOF eyes, expression of an enhancer-trapped lacZ at the glass locus is greatly reduced [2964]. dPax2 cut. Expression of cut vanishes temporarily in cone cells in dPax2 LOF eyes [1320]. dPax2 Bar-C. Expression of Bar-C genes disappears in R1/6p cells in dPax2 LOF eyes [1320]. Mδ BarH1. Expression of BarH1 vanishes in R1/6p cells under mδ GOF conditions (sev-Gal4:UAS-mδ), while R7p expresses BarH1 under N LOF (mδ LOF ) conditions [870].

The links among these transcription factors (excluding autoregulatory loops) are presented as a wiring diagram in Figure 7.9b, and their R-cell expression patterns are cataloged in Table 7.1. How the circuit operates is unclear. There is no obvious “receptor code” wherein each R subtype (e.g., R1/6) is encoded by a particular set of transcription factors. The only hint of such a code is the aforementioned enforcement of outer-type R-cell identity by Rough or Svp. Undoubtedly, there are important links missing, and further screens will be necessary to

220 TABLE 7.1. CELL-TYPE SPECIFIC EXPRESSION OF BOSS, SEVENLESS, AND GENES THAT ENCODE TRANSCRIPTION FACTORS IN OMMATIDIA*

Gene

R8p Cell (Inner)

R2/5p Cells (Outer)

R3/4p Cells (Outer)

R1/6p Cells (Outer)

R7p Cell (Inner)

Cone Cells

boss (ligand) [4890] .

ON & required.

—

—

—

—

LOF: R7 (sic) becomes cone cell. — GOF: fails to develop?

— GOF: fail to develop?

ON but not

ON (weakly) but

ON & required.

—

—

—

—

—

ON & required. LOF: no obvious fate switch.

—

sev (receptor for Boss) [604, 1049, 1677, 4363] .

atonal {bHLH} [4621].

Bar-C {HD} genes (H1 and H2) [1768] .

ON & required. LOF: usually missing (no further cells recruited). —

needed.

not needed.

LOF: becomes cone cell (but # of cone cells remains 4). —

PPC, SPC, TPC Cells

Bristle Cells

— GOF: become R7s.

—

—

ON but not needed. GOF: become R7s.

— GOF: fail to develop.

—

—

—

—

ON & required in

ON & probably

PPCs. LOF: no obvious fate switch.

required in neuron and sheath cells.

—

—

—

—

—

— GOF: become outer-type R cells (R1/6?) or PPCs or disappear. ON.

—

—

—

ON.

ON.

ON & required. LOF: missing?

ON & required. LOF: missing?

dPax2 {paired & ∼HD} [1256, 1319, 1320].

—

—

ON & required. LOF: missing (infrequently). —

ON & required. LOF: missing (infrequently). —

ON & required. LOF: missing (infrequently). —

eyes absent (eya) {?} [397, 930]. glass {ZF} [1157,

ON.

ON.

ON.

ON.

ON.

?

ON & required. LOF: arises but dies before maturing.

ON & required. LOF: arise but die before maturing.

ON & required. LOF: arise but die before maturing.

ON & required. LOF: arise but die before maturing.

ON & required. LOF: arises but dies before maturing.

ON (weakly) but not

cut {HD} [373, 3659] . dachshund {?} [870]. dJun {bZip} [2280, 2281] .

2963, 2964, 3161, 4391].

—

ON.

—

—

—

ON. GOF: become R7s.

—

—

ON & required. LOF: arise but abn. diff.

ON & required in

ON in all cells of

PPCs. LOF: arise but fail to form the lens properly.

the bristle organ. LOF: arise but are malformed and misplaced. ?

?

needed.

ON but not

ON but not

needed.

needed.

221 lozenge {runt} [241, 897, 938, 1255].

—

—

— GOF: become R7s.

ON & required. LOF: absent or abn. dev.

ON & required. LOF: becomes outer-type R cell.

mδ {bHLH} [870, 1271].

—

—

ON in R4.

—

muscleblind {ZF} [265].

ON & required. LOF: arises but abn. diff.

ON & required. LOF: arise but abn. diff.

ON & required. LOF: arise but abn. diff.

ON & required. LOF: arise but abn. diff.

ON & required. LOF: becomes outer-type R cell. ON & required. LOF: arises but abn. diff.

onecut {cut & HD} [3107].

ON & required. LOF: arises but abn. diff. ON & required. LOF: arises but abn. diff. —

ON & required. LOF: arise but abn. diff. ON & required. LOF: arise but abn. diff. —

ON & required. LOF: arise but abn. diff. ON & required. LOF: arise but abn. diff. —

pipsqueak (BTB-domain cofactor for Svp?) [4567]. pointed {Ets} [516, 3162, 4381].

—

—

ON & required.

ON & required. LOF: arise but abn. diff. ON & required. LOF: arise but abn. diff. ON & required. LOF: become cone cells. —

ON & required. LOF: arises but abn. diff. ON & required. LOF: arises but abn. diff. ON & required. LOF: becomes cone cell. —

ON & required. LOF: fails to arise.

ON & required. LOF: fail to arise.

ON & required. LOF: fail to arise.

ON & required. LOF: fail to arise (easily affected).

prospero {∼HD}

—

—

—

—

ON & required. LOF: fails to arise (easily affected). GOF: becomes cone cell (in yan-LOF background). ON (at higher level than cone cells) & required for repression of R8-type rhod.

orthodenticle {HD} [4461]. phyllopod {?} [717, 1052, 4249].

[1070, 1753, 2165, 4775].

ON & required. LOF (weak): become R7s. Null: become outer-type R cells. —

ON.

—

—

—

ON (weakly) but not

ON (weakly) but

ON (weakly) but

needed. LOF: suppresses transf. to R7s (or other R cells) caused by svp-GOF. —

not needed.

not needed.

—

ON in neuron.

—

—

—

— GOF: become R7s. —

— GOF: become neuronal. —

—

ON but not needed?

ON but not

ON but not

needed?

needed?

—

LOF: absent.

ON.

—

—

(continued)

222 TABLE 7.1 (continued )

Gene

R8p Cell (Inner)

rough {HD} [232,

—

1676, 1783, 2223, 3723, 4361, 4459] .

senseless (sens) {ZF} [1279, 3127].

seven in absentia (sina) {ZF} [671].

ON & required. LOF: becomes R2/5. GOF: extra R8s. —

R2/5p Cells (Outer)

R3/4p Cells (Outer)

R1/6p Cells (Outer)

R7p Cell (Inner)

Cone Cells

PPC, SPC, TPC Cells

Bristle Cells

ON & required. LOF: lose R2/5 identity and mimic R3/4 or R1/6. R3/4 & R1/6 dev. abn., but R7 is ∼normal, and an extra R7 often arises. —

ON but not

—

— GOF: becomes outer-type R cell.

—

—

—

—

—

— LOF: absent.

—

—

—

—

ON but not

ON but not

ON & required. LOF: becomes cone cell. — Extreme GOF: becomes outer-type R cell.

ON but not needed.

—

—

needed (except at late stage?). ON & required. Null: become R7s.

— Mild GOF: become R7s. Extreme GOF: become outertype R cells.

—

—

ON & required? LOF: dies. ON but not needed?

ON & required? LOF: dies. —

ON & required? LOF: dies. —

ON (p69 & p88) &

ON (p69 & p88).

ON (p69 & p88)

needed.

seven-up (svp) {ZF} [264, 1856, 2322, 2888].

—

— GOF: remain neuronal but lose R2/5 traits.

sine oculis (so) {HD} [759, 930]. spalt {ZF} [220, 221, 2902, 2903, 4855] .

ON & required. LOF: dies. ON & required (late). LOF for Spalt-C: makes R8-like axon. proj., but ≈ outer-type R cell in rhabd. (large) & rhod. (Rh1). ON (p69 only) & required (late). LOF (p69): arises but fails to complete dev. GOF (p69 or p88): fails to arise (or dies thereafter).

ON & required. LOF: dies. ON (weakly?) but not needed.

needed (except at late stage?). ON & required. Null: become R7s. An extra outertype R cell arises when R3 & R4 are both svp-null. ON & required. LOF: dies. ON but not needed.

ON (p69 only) &

ON (p69 only) &

ON (p69 only) &

required (late). LOF (p69): arise but fail to complete dev. GOF (p69 or p88): fail to arise (or die thereafter).

required (late). LOF (p69): arise but fail to complete dev. GOF (p69 or p88): fail to arise (or die thereafter).

required (late). LOF (p69): arise but fail to complete dev. GOF (p69 or p88): fail to arise (or die thereafter).

tramtrack {ZF} [670, 2391,2529, 4773] . Isoforms:

p69 (activator or repressor?) and p88 (repressor).

ON & required. LOF: dies. ON (weakly?) but not needed.

ON & required. LOF: dies. ON & required (late). LOF for Spalt-C: makes R7-like axon. proj., but ≈ outer-type R cell in rhabd. (large) & rhod. (Rh1). ON (p69 only) & required (late). LOF (p69): arises but fails to complete dev. GOF (p69 or p88): fails to arise (or dies thereafter).

required. LOF: become R7s (?) or fail to develop. GOF (p69 only): absent.

in shaft, socket, and sheath cells; p69 is required in IIa and sheath cells (cf. App. 3).

223 ultraspiracle {ZF} [3198, 4855]. yan {Ets} [2392, 3162, 3467, 4791] .

ON & required. LOF: arises but abn. diff. ON (transient?). LOF: fails to arise (re-enters cell cycle). GOF (activated construct): lost.

ON & required. LOF: arise but abn. diff. ON (transient?). LOF: fail to arise (re-enters cell cycle). GOF (activated construct): lost.

ON & required. LOF: arise but abn. diff. ON (transient?). LOF: fail to arise (re-enters cell cycle). GOF (activated construct): lost.

ON & required. LOF: arise but abn. diff. ON (transient?). LOF: fail to arise (re-enters cell cycle). GOF (activated construct): lost.

ON & required. LOF: arises but abn. diff. ON (transient?). LOF: fails to arise (re-enters cell cycle). GOF (activated construct): lost.

—

—

—

ON (weakly). LOF: become R7s? GOF (activated construct): fail to arise.

ON.

ON in one cell

only.

∗ “ON” means that the gene is transcribed in the indicated cell type; “—” means that no expression has been detected (or reported). Motifs (DNA-binding or other) are in braces, and roles (in signaling) are in parentheses. Domain abbreviations: HD (homeodomain), ZF (zinc ﬁnger). Phenotype abbreviations: abn. (abnormal), axon. proj. (axonal projection), dev. (development), diff. (differentiation), rhab. (rhabdomere), rhod. (rhodopsin), transf. (transformation). Gene abbreviations: boss (bride of sevenless), sev (sevenless). Cell types are listed left to right in order of their appearance (cf. Fig. 7.6). PPC, SPC, and TPC (Primary, Secondary, and Tertiary Pigment Cells) are distinct types but are grouped here to conserve space. The “p” sufﬁx denotes a precursor cell, and slash marks indicate pairs of histologically similar cells (e.g., R1/6p = R1p and R6p). Considering the bristle as a single cell (the SOP generates a neuron, sheath, shaft, socket, and glial cell; cf. Fig. 2.1), but not counting R3-vs.-R4 asymmetry (cf. Fig. 7.5), the ommatidium has 10 basic cell types. LOF and GOF designate loss- or gain-of-function effects. The so-called “mystery cells” (not listed) afﬁliate transiently with nascent clusters [186, 4364, 4715] and are known to express sev [225, 1049] and dJun (weakly after R2/5/8p and before R3/4p) [383]. These few cells are converted to photoreceptors by argos LOF [1294], boss GOF [225, 1049], echinoid LOF [170], fat facets LOF [746, 748, 1241,1932], groucho LOF [1241], liquid facets LOF [594], rhomboid GOF [1293], sev GOF [225, 229, 1049], yan LOF [2392], and probably also by Delta LOF [3273], extramacrochaetae LOF [508], PLCγ LOF [2669], pointed GOF [516], spitz GOF [1289], string LOF [2971], tramtrack LOF [4773], and ultraspiracle LOF [457, 4855], based on extra-R7 phenotypes. Mystery cells are normally blocked from adopting an R-cell fate by an unknown signal from neighboring (non-R) cells [594, 1241,1932]. Transmission of the signal appears to require speciﬁc intercellular adhesive contacts [170, 2492, 3106] and speciﬁc phasing of the cell cycle [747]. In the honey bee Apis mellifera, a second R7-type (UV-sensitive) cell apparently arises from a mystery cell via a contact with R8p that fruit ﬂies lack [1049, 1149, 3538]. Several of these genes were named for effects on eye shape (Bar, lozenge), texture (glass, rough, scabrous), or R-cell composition (boss, sevenless, seven-up) [2561, 4357]. The transcription factors Eyelid (Bright family of DNA-binding domains) [4389], Eye gone (paired-HD) [2090, 2099], and Tailless (zinc-ﬁnger class) [955] are expressed in eye cells, but their roles are unclear. The same is true for the co-activators Blind spot and Kohtalo [4386]. Certain genes that do not encode transcription factors also regulate the differentiation of photoreceptors (e.g., chaoptic [2323, 3568, 4458] and the Broad-Complex isoforms [457]) or neurons in general (e.g., abl [289], deadpan [331,1164], elav [518, 938, 1887, 2312, 4805], and scratch [1164, 3611]). Other genes are expressed or needed in subsets of photoreceptors [4600] – e.g., dachshund (R1/6 and R7) [2689], daughterless (R8, R2/5, and R3/4) [506], Delta (intense in R3/4 and R1/6) [870], dead ringer (R8 and R1–R6) [3917], hairy (expressed in R7 but not needed) [507], hedgehog (strongly in R2/5, weakly in R8, and later in all) [1077, 2632, 4621], scabrous (R8 only) [179, 182,186, 897, 2461], retina aberrant in pattern (R8 only) [2145, 3390], rhomboid (R8 and R2/5) [897, 4035], semang (R1/6 and R7) [4869], Star (R8 and R2/5) [1782, 1785], vein (R8 only) [4035], and the paralogs Brother and Big brother (needed for lozenge function in R1/6 and R7) [2524]. Cone cells speciﬁcally require rugose [4470]. This table is an expanded version of tables in [1047, 2351, 2961]. Genes whose products function in the Sev pathway between the receptor and its nuclear effectors are omitted. Their LOF-GOF effects are as expected, given the positive or negative roles of their products (cf. Fig. 6.12) [264, 1048, 4790] – e.g., Ras1 [1273] and dMAPK [517]. Also omitted are genes in the Notch pathway (e.g., mδ in R4 [869]), which show transient expression in various precursors. See [897, 3574] for more cell-type markers and [1293, 2887, 2903] for enhancer-trap lines that express lacZ in subsets of R cells. See [1161, 3708] for spalt’s role in enforcing alternative cell identities in another PNS context. See [4052] for evolution of opsins and [968, 1272, 3895] for evolution of upstream circuitry. N.B.: Ectopic overexpression of scute rescues ato LOF eyes without restoring discernable R8 (or R7) cells [4209]. Conceivably, scute is enabling outer R cells to bypass their need to be induced by R8p, but a more prosaic explanation is that the mutant R8p progresses far enough in its maturation to induce R1–6. Indeed, boss-on R8p cells do arise in scute GOF eyes, although their development is delayed and their numbers are reduced. The same explanation may hold for senseless LOF eyes, whose ommatidia develop without mature R8 (or R7) cells [1279]. In the latter eyes, R8p cells do not express boss or scabrous, but they do express atonal faintly. Remarkably, R8 (boss-on) cells are rescued in senseless LOF rough LOF double mutants (attributable to a rough” link) [1279]. Multiple R8 cells per ommatidium arise in shattered LOF eyes [4247]. “senseless

224

ﬁnd the genes that can solve the puzzle. Fortunately, ingenious new screens are now underway [3381, 4132, 4292]. One of the many related riddles concerns tramtrack (ttk) [1051], which encodes two zinc-ﬁnger transcription factors (cf. Fig. 2.5). The p88 isoform prevents neural development wherever it is expressed [670, 2529, 4249]. Ttk88 is targeted for proteolysis when it binds Phyl and Sina (cf. Ch. 6) [4249, 4382]. Both Phyl and Sina are normally expressed in R1/6p and R7p cells, so it is easy to see how these cells become neural. What is not obvious, however, is how the other precursors (R8p, R2/5p, R3/4p) manage to stiﬂe Ttk88 without the Phyl-Sina duo. Phyl has its own secrets [1052]: phyl is supposed to be transcriptionally upregulated by the Ras-MAPK pathway [717, 1047, 1052, 4381], and this pathway is activated in all R cells [670, 1289, 1337], but phyl is only expressed in a subset thereof (R1/6/7p) [717, 1052]. Why? Recently, two key clues to the Photoreceptor Coding Enigma have come to light. First, two teams (Cooper and Bray [870] and Tomlinson and Struhl [4367]) showed that R7p requires a Delta signal from R1/6p in addition to a Boss signal from R8p. This discovery has revived the Combinatorial Cascade Model, at least with regard to R7: 1. Removing Delta from R1p and R6p causes R7p to develop as an R1/6-like photoreceptor, whereas disabling Delta in R8p has no such effect. 2. Suppressing the Notch pathway in R7p (via a chimeric Su(H)-Engrailed repressor) likewise causes it to adopt an R1/6-like fate. 3. Activating the Notch pathway in R1/6p cells induces them to become R7s. Second, Mollereau et al. reported that both R7 and R8 cells become outer-type (R1–R6) photoreceptors in Spalt-Cnull eyes, although axonal projections are unchanged [2902]. Expression of spalt differs in early (R1p– R6p) vs. late (R7p–R8p) eye development, and this shift may signify a redeployment of spalt to encode certain aspects of the differentiated state (i.e., rhabdomere size and rhodopsin subtype). Prospero likewise suppresses R8-type rhodopsins in the R7 cell by directly binding the rh5 and rh6 cis-enhancers [860].

The lattice is created by inhibitory fields around R8 precursors For anyone interested in pattern formation the abiding allure of the ﬂy eye has always been its “Lattice Riddle”: how is the ommatidial lattice created? Ready et al.’s

IMAGINAL DISCS

Crystallization Model was a good ﬁrst guess, but, as discussed above, it proved false. We now know that newly recruited cells do not “read” their fates from their predecessors. Rather, new cells assemble in shells around each R8p founder, with no interommatidial communication after the R8p “seed” is planted [4769]. The remaining question, of course, is how those seeds get planted so neatly in the ﬁrst place. Because the lattice acquires “nodes” (R8 sites) sequentially, old nodes could be dictating where new nodes arise. A phenomenon of this sort was described in hemipteran insects by Wigglesworth in 1940: bristles that arise in later instars are constrained to certain sites by bristles that arose in earlier instars [4657]. The Lattice Riddle was essentially solved in 1972 by Donald Ede [1140] – a disciple of the great theorist C. H. Waddington [1486, 4826]. Ede was familiar with Wigglesworth’s work, and he was adept at building computer models. He combined Wigglesworth’s notion of node-centered inhibitory ﬁelds [4657] with the concept of a node-inducing wavefront to craft the “Inhibitory Field and Wavefront Model” [1140]. This scheme was actually designed to explain feather lattices in bird skin, but it works equally well for the ﬂy eye. The main feature that Ede was trying to simulate is the 0.5 internode offset between adjacent columns – an offset that automatically tessellates the plane with equilateral triangles (Fig. 7.7). (Italics are author’s.) One most tantalizing system which appears ripe for this sort of analysis . . . is the establishment of the beautifully regular diamond lattice pattern of feather papillae on the back of the chick embryo. . . . The condensations which give rise to feather papillae are initiated in single files, the first of which is immediately over the embryonic neural tube and vertebral column, and the back on either side is subsequently covered by condensations which appear file by file, rather like a line print-out from a computer. Experimental embryologists have shown that production of the feather papillae is dependent on induction by the neural tube and/or the vertebral column, that is the mesenchyme on either side of these structures only becomes capable of producing condensations as some substance diffuses outwards from the midline on each side. Thus, as a model, we may suppose that at first only a narrow band of skin is capable of undergoing mesenchymal condensation, or of initiating this process, but that this band becomes gradually broader. . . . As each condensation is produced a zone of inhibition is generated around it, and these zones will intersect at the apices of equilateral triangles whose bases extend from one condensation to the next. As soon as the induced band has extended to these points, the next file of papillae will be initiated. In this way, as one file is produced after another, the lattice arrangement of feather papillae is marked out. [1140]

CHAPTER SEVEN. THE EYE DISC

A similar argument was made by Patricia Renfranz and Seymour Benzer in 1989 [3574]. Like Ede, they stressed the half-phase offset between columns, but instead of a wave per se, they proposed that inductive signals (as well as inhibitory ones) emanate from the nodes (preclusters) themselves. In the Drosophila eye disc at the morphogenetic furrow, negative and positive feedback loops between neighboring cells, like those acting in embryonic neurogenesis (reviewed in [626]), could control the spacing between nascent ommatidia at the furrow. Mathematical models of biological pattern formation, particularly formation of periodic structures, often invoke diffusible factors. These models postulate the presence of an activator and an inhibitor, the inhibitor diffusing more readily than the activator (for review, see [2806]). Note that, in each successive column, the clusters are on diagonal centers, rather than immediately behind those in the preceding column. An activator of cluster formation, coupled with an inhibitor, could provide a mechanism for regulating the spacing, and therefore the number, of ommatidia in a column. In the differentiating eye disc, such a mechanism would likely involve the cells that form ‘‘nucleation sites’’ at which the nascent ommatidia condense as five-cell preclusters at the furrow. Lateral inhibition between nucleation sites could control the number of ommatidia seeded in a column, and the activator could, in turn, influence the number of cells in each cluster. Such a mechanism, involving graded inhibitory signals, has been suggested for the regular spacing of sensory bristles on the insect cuticle [2959, 4657]. [3574]

In 1990, Nicholas Baker, Marek Mlodzik, and Gerald Rubin [179, 2885] proposed that the inhibitor is Scabrous (Sca) – a secreted glycoprotein that is distantly related to vertebrate ﬁbrinogen [1921, 2461, 2463]. Sca is secreted by R8p cells at the trailing edge of the MF, and the forward diffusion of this molecule could easily constrain the next row of sca-on cells via a “Sca sca” feedback loop. . . . the most straightforward model is that the sca protein itself is an extracellular regulator of ommatidial spacing. [179] The observed eye disc phenotype indicates that sca plays a role in establishing the initial periodicity, because the spacing of preclusters and, thus, R8 precursors, in mutant eye discs is clearly irregular. Potentially, there may be interactions between forming preclusters and the assembled column immediately posterior. Indeed, the precise positioning of each new row one-half unit out of phase with respect to the last suggests that the existing pattern has an influence and that sca is involved in this regulation, as judged from its phenotype. Inhibition governed by sca expression in the R8 precursor might extend from the next posterior column, inhibiting sca expression in cells that are closer to the already developing precluster, and thus lead to the observed very regular array of developing ommatidia. [2885]

225

Sca has been thought to control not only the spacing of ommatidia in the eye but also the spacing of bristles throughout the entire body (cf. Ch. 3). Indeed, these two processes share other key features in common [229, 1804, 4621]: Bristle Spacing

Ommatidial Spacing

1. Sca is transiently expressed in all cells of the proneural cluster (PNC), and it persists in the sensory organ precursor (SOP) [2885].

1. Sca is transiently expressed in “intermediate groups” [2042] (although this step is dispensable [1077]), and it persists in the R8p [179, 183, 186, 2461].

2. For a cell to become a SOP, it must express the ubiquitous proneural gene daughterless [905, 949, 4452] as well as the “regional” proneural genes achaete or scute [912, 3982].

2. For a cell to become an R8p,itmustexpressdaughterless [506] as well as the “regional” proneural gene atonal [2041, 2042].

3. The PNC is narrowed to a single SOP via the Notch pathway, using the ligand Delta [3022, 3270] but not Serrate [4859]. If that pathway is disabled, then many cells in the PNC become bristles [112, 3022].

3. The “R8 equivalence group” (i.e., the group of atonal–on cells) is narrowed to one R8p via the Notch pathway using the ligand Delta [3273] but not Serrate [183, 2549]. If that pathway is disabled, then most cells in the group become R8s [179, 184, 603, 2461, 2549, 3273] (cf. a similar circuit in mice [4354]). Curiously, R1p-R7p can become neural without expressing atonal or any other bHLH factor [1616].

In the above table, the terms “intermediate group” and “R8 equivalence group” are used synonomously [184]. These groups of ∼12 cells (henceforth called “IGs”) express both sca [179, 1158, 1921] and the proneural gene atonal (ato) [2042, 4621]. They are spaced at uniform intervals near the front of the MF. Since they immediately precede the rosettes (column 0), they are considered to occupy column “−1” [184, 2042]. These nests of sca-on-atoon cells dwindle to single R8p cells by column 0. If Sca is responsible for ommatidial spacing, then its secretion by R8p cells (column 0) must constrain new sca-on cells in IGs (column −1). The constraint may even happen earlier [186, 2461] because groups of ato-on cells (“initial clusters”) have been detected in column “−2” [1077, 4208]. The main evidence for Sca as the R8p inhibitor is the sca null phenotype. Incipient ommatidia are closer together in sca null eyes [179]. However, the crowding is milder than it should be if the inhibitory mechanism

226

IMAGINAL DISCS

MF x: R2/5p y: R3/4p z: R1/6p

D

x 8 x

P

A V

d!

Key:

C Gr on ad ica ien l t

a

Inhibitor: Argos? Scabrous?

w N o R 8 s a ll o

e

MF

8 x 8 x

b Inhibitory fields

8 x 8 x

t

~1.5 h

8

c

y x 8 y x y x 8 y x y x 8 y x

z y x 8 y x z z y x 8 y x z

z y x 8 7 y x z z y x 8 7 y x z z y x 8 7 y x z

z y x 8 7 y x z z y x 8 7 y x z

z y x 8 7 y x z z y x 8 7 y x z z y x 8 7 y x z

MF x 8 x 8 x 8 x 8 x 8 x

y x 8 y x y x 8 y x

z y x 8 y x z z y x 8 y x z z y x 8 y x z

CHAPTER SEVEN. THE EYE DISC

were totally disabled (cf. stronger NLOF and Dl LOF phenotypes [179, 184, 186, 603, 2461, 3273]): only rarely do R8p cells emerge as doublets (or higher multiples) from the MF [179, 186, 1158]. The implication is that sca acts redundantly with some other gene [179]. Genetic interactions between sca and Notch [1921, 2462, 2885, 3490] suggested that Sca might use Notch as its receptor [179], but Sca does not bind Notch [2460]. The actual Sca receptor remains unknown [178, 2461]. The main snag with the idea of Sca as the R8p inhibitor is the sca GOF phenotype [186]. Overexpressing Sca at the front of the MF should extinguish ommatidia, but in fact, it causes ommatidial crowding just like sca LOF [1158]. The LOF and GOF phenotypes also resemble one another insofar as nascent ommatidia are more irregularly spaced. This disorder suggests that cells default to a more random Delta-Notch contest when the amount of Sca is too low or too high [1158]. Conceivably, Sca biases these contests. R8p cells might arise wherever cells sense a particular slope (vs. absolute value) in Sca concentration. The ideal slope would exist near the edge of Sca’s diffusion range, although it is unclear whether (and, if so, how) cells can measure such slopes [2434, 2448]. Unraveling this circuitry has been complicated because Notch is used twice during the process [182, 183, ato) and 2549, 3028]: ﬁrst to activate ato in rosettes (N later to suppress ato in all cells except R8p (N ato). Sca apparently participates in the ﬁrst phase but not the second [186]. (At the ato locus, there are separate cisenhancers for expression in the rosettes vs. in the fainter band that precedes them [4208].)

227

In 1998, Spencer and Cagan argued that the diffusible R8p inhibitor is Argos, not Sca [4035]. They suspected the EGFR pathway in the bestowing of R8 competence because high levels of Argos [4035], Spitz [4322], and activated dMAPK [2355, 2493, 4035] are detected with Atonal [184, 4035] in the same 6–10 cells in each rosette. However, the EGFR pathway is not needed for ommatidial spacing: spacing is nearly normal in Egfr null [2355], rhomboid null [4035], and spitz null [1083, 4322] clones (although R8 cells fail to form inside doubly mutant rhomboid null vein null clones [4035]). Null alleles of argos do alter the lattice, but their effects are mild [204, 1294, 4035]. The absence of a strong LOF effect does not rule out Argos as the inhibitor since it might have a redundant partner. However, the hypothesis seems untenable in the face of a more troubling fact: argos LOF eyes can be restored to a wild-type phenotype by expressing argos with a sev enhancer [1287]. How important can Argos be for R8p spacing if it can be erased from the rosettes (by argos LOF ) and banished to clusters behind the MF (by sevE-argos) without disturbing the R8p pattern? Thus, neither Scabrous nor Argos can claim “hero” status in this saga [4621]. The Lattice Riddle therefore endures as a daunting challenge in search of a molecular protagonist [204].

The lattice is tightened when excess cells die Given that ommatidia self-assemble as separate islands by recruiting cells from the surrounding epithelium, the eye encounters a serious engineering problem. If the R8p founder cells are spaced too close together, then

FIGURE 7.7. The Inhibitory Field and Wavefront Model for creating a hexagonal lattice. See also Appendix 7.

a. Mature left eye disc. Vertical stripe is the morphogenetic furrow (MF). Compass (at right) gives axes in the fate map (cf. Fig. 7.1). The MF leaves a hexagonal lattice of ommatidial cell clusters (dots) in its wake. Oval (far right) denotes an inhibitory ﬁeld wherein no R8 photoreceptors can arise. This ﬁeld is supposed to be generated by a pre-existing R8 precursor (“8” or “R8p”) and possibly other R-cell precursors (e.g., “x” = R2/5p) [2493]. The identity of the secreted inhibitor is unknown. Suspected candidates have included Argos [4035] and Scabrous (Sca) [179, 2352, 2885], but Argos cannot be acting alone because argos null clones have normal spacing of R8s [204]. Secretion implies a cone-shaped gradient. b, c. Imaginary slices of the columnar epithelium (magniﬁed from box in a) at two times separated by ∼1.5 h (“t”). The MF is a groove (black) that runs along the D-V axis. It moves from P to A (long arrows). R8 precursor cells (teardrop shapes) arise within “intermediate groups” (not shown; cf. Fig. 7.9) at intervals along the MF [184] and found the ommatidia (cf. Fig. 7.6) [1048, 1279]. Gray stripes mark columns of nascent ommatidia, which are older (and hence more complete) as distance (and time) from the MF increases. Successive columns are out of register (shifted vertically) by 0.5 R8p interval, thus forming a hexagonal lattice. The phase shift is attributable to rules that govern how new R8p cells arise: for a cell to become an R8p, it must be (1) in the MF and (2) not in an inhibitory ﬁeld. Thus, as the MF moves anteriorly (compare b with c), the ﬁrst cells that become competent for the R8 fate are those that lie in the crevices of the preceding inhibitory ﬁelds. Fields must arise within ∼1.5 h after R8p inception. How long they persist is not known. Here they are shown in only one column, although expression of Sca (and Ato) persists in R8p cells for ∼4 columns [183, 1158, 4621]. Also unknown is whether the signals come from single R8p cells [2885], from nests of a few cells as shown here [2632, 4035], or from the intermediate groups whence R8p cells arise [186, 2461, 4208]. The signaling cells are presumably deaf to the inhibitor because they probably disable their receptors (or transduction pathway) before they start signaling.

228

the epithelium will run out of cells before every ommatidium acquires its quota [1003, 3655]. However, if R8p cells are too far apart, then lots of unrecruited cells will be left over [2493]. Stationing R8p cells at exactly the right intervals ab initio is not an option because the spacing mechanism uses a sloppy diffusible signal. Evolution solved this “Spacing Precision Problem” by adding a “clean-up” phase after the spacing and accretion phases have run their course (cf. the “ﬁne-tuning” tricks in bristle patterning; e.g., Fig. 3.9d). Thus, more cells are allocated to the epithelium than will be recruited, and the excess cells are pruned by cell death [602, 3509, 4715]. This “Kill the Stragglers! Trick” of producing an excess of competent cells and then destroying those that do not get incorporated is common in animal development [2009, 2840, 3493, 4037] – especially in nervous systems [887, 888, 1141, 3187, 3189, 3358] where it ensures parity of interconnected populations [798, 2149, 2150, 3188, 3478, 4685], as in the ﬂy’s retinolaminar projection [624, 1270, 4774]. At the beginning of pupation, the retina has more cells than it will ultimately use. As the initially disordered interommatidial cells are sorted into a lattice, ∼2000 excess cells are eliminated by cell death. . . . Cell death appears to eliminate cells which fail to establish contacts appropriate to a specific cell type. As interommatidial cells are sorted into the lattice, some cells are left with no opportunity to make the contacts needed to survive. For example, a cell may be blocked from contacting a second primary pigment cell by the presence of an intervening cell. The blocked cell will be eliminated by cell death. As surplus cells are eliminated, the lattice tightens. At this stage two cells are often equally positioned to become the single secondary seen in the adult. The later phase of cell death generally eliminates one of these cells. [602]

This “Darwinian” strategy for tightening the lattice is clever, but it puts the eye in a precarious situation. To wit, slight mistakes in the timing or strength of induction could lead to runaway cell death. This instability may help explain why so many genes can mutate to give a small- or no-eye phenotype [396, 2561]. Other consequences of the strategy are seen in various features of eye development: 1. Rosettes in the MF are spaced more irregularly than the nodes in older columns [184, 4715]. 2. The extra interommatidial cells can replace missing cone cells when the latter are forced by sev GOF to become R7s [225]. (Their transition to a cone-cell identity may be too late for sev GOF to also turn them into R7s.) 3. The number of deaths varies among normal ommatidia, as expected if there is “noise” in the initial spac-

IMAGINAL DISCS

ing of R8p cells [4769]. The average loss is 2 to 3 cells per ommatidium [4713]. 4. More extensive cell death is observed in Egfr GOF discs whose clusters are widely separated [181], and the death occurs earlier (columns ∼10–15 vs. a broad swath behind column 12 [4715]). Stragglers commit suicide via the usual pathway of apoptosis (a.k.a. programmed cell death) [201, 1590, 2774, 2797, 4618]. In ﬂies, this pathway is executed by grim [741, 3621], reaper [3134, 3621, 4078, 4619, 4620], and hid (head involution defective) [1621, 4402] via the caspases (cysteine proteases) DCP-1 [4018], drICE [1283], Dredd [742], etc. [1941]. The Notch pathway is also involved [396, 603, 605, 2859, 3273, 4248]. Doomed cells can be rescued by Egfr GOF , Ras1 GOF , or argos LOF [515, 2859]. This result chimes nicely with the Stop the Clock! Model (see above) [2859], if one assumes that the ﬁnal tick of the clock causes a cell to kill itself. Spitz would thus be a survival factor in addition to its fate-assigning role [185]. In this scenario, each epithelial cell would be brooding: “If Spitz does not arrive before midnight, then I’ll kill myself!” Consistent with this surmise, more cells die when the Spitz signal is blocked in argos GOF or Egfr LOF eyes [201, 1287] or in Star null clones [1785, 1788]. Likewise, extensive apoptosis occurs anterior to the MF in “furrow-stop” mutants where the MF halts prematurely (Bar-CGOF [1309, 1310, 1788], dpp LOF [724], Drop LOF [1788, 2971], hh LOF [406, 724, 1788, 2632], rough GOF [725, 1788], and possibly daughterless LOF [506]) as well as in mutants where the MF never initiates properly (dachshund LOF [2689], dpp LOF [529, 2739], eyeless LOF [1309, 1684], eyes absent LOF [397, 2485], and sine oculis LOF [759] but apparently not atonal LOF [2042]). Finally, Spitz’s rescuer role is conﬁrmed by the nonautonomy of apoptosis in spitz LOF clones: core cells die, but cells near the edge survive apparently due to Spitz diffusing in from surrounding wild-type cells [185]. The level of EGFR signaling needed to keep a cell alive is less than the level needed to change its fate [1687]. Anterior cells can be rescued by agents that block apoptosis, whereupon they become SPCs or TPCs [1763, 4248]. Evidently, SPC and TPC are the default states when suicide is not an option.

Eye bristles arise independently of ommatidia Apoptosis occurs maximally between 35 and 55 h AP (at 20◦ C) in wild-type eye discs [515, 1287, 1289, 4713, 4715]. By the start of this period, ommatidia have acquired all their photoreceptors (8), cone cells (4), and PPCs (2) but have not yet conscripted SPCs, TPCs, or bristle cells

CHAPTER SEVEN. THE EYE DISC

from the disorderly background of interommatidial cells [602, 4713]. Bristle development is well advanced by this time because (1) ac-on proneural clusters appear just after pupariation [597], (2) bristle SOPs arise at ∼12–24 h AP (20◦ C) [3273], and (3) differentiative mitoses ensue at ∼14– 28 h AP (20◦ C) [602, 1287, 4715]. Virtually every background cell can be forced to make a bristle (at the expense of SPCs and TPCs) by heating t.s. Dl LOF mutants at 11–18 h AP (20◦ C) [3273] (cf. N LOF [603]). This phenotype indicates that the Notch pathway selects bristle cells within the eye ﬁeld, just as it does in other discs (cf. Ch. 3). Because the same pathway also helps assemble ommatidial clusters [603], there is the potential for chaotic cross-talk between SOPs and R cells, which could wreak havoc on the lattice [1804]. The ﬂy avoids such chaos by using the Heterochronic Superposition Trick (cf. Ch. 3). It times its SOP selection and R-cell recruitment so that these periods do not overlap. Indeed, SOPs do not rely on the MF at all [597]. They develop in a separate wave that spreads radially from the center to the periphery, not from P to A [602, 603, 605, 4715]. Moreover, bristles avoid using the EGFR pathway: argos LOF eyes have extra R cells, cone cells, and pigment cells, but the number of bristles (albeit disorganized) is normal [1294]. Bristles assume their ﬁnal positions at ∼35–40 h AP (20◦ C), as the lacework of background cells “shrink wraps” each ommatidium via apoptosis [4713]. When bristle initiation is prevented throughout the eye (by wg GOF ), the facet vertices where bristles would have sprouted are occupied by extra TPCs [597]. Because eye bristles arise independently of ommatidia, it is not surprising that the vertices to which they are relegated are not as orderly as the ommatidial lattice itself. These vagaries are apparent in the wild-type eye schematized in Figure 7.8. Most bristles reside at the anterior end of the horizontal edge between facets, but there are many exceptions to this trend [3539]. The exceptions obey a rule of their own: whenever a bristle is missing from the A end of the edge, a bristle is found at the P end (except at the periphery, which is bald). These A-to-P displacements are common in the anterior third of the eye but are sporadic elsewhere. Why are bristles near the front of the eye less choosy? One clue to this “Fickle Bristle Mystery” (cf. Fig. 5.12g) comes from the eyes of t.s. N LOF mutants that were heat-pulsed during the sensitive period for extra eye bristles. About 6 columns behind the vertical extrabristle “scar,” ≥30% of the bristles are shifted to the P end of the edge between facets [603]. Apparently, the

229

operational proneural cluster (PNC) stretches along each horizontal edge (SPC cell) [1804]. Within this PNC, only one SOP normally originates. Throughout most of the eye, the SOP might arise at the PNC’s A end due to a “prepattern” agent that biases the Delta-Notch competition in that direction (cf. Fig. 3.6). If that agent is linked to the MF, then the ﬁckleness of the anterior region could have something to do with the fact that the MF travels through this part of the eye ﬁeld after pupariation [602, 1287, 4715]. During that period, the MF moves faster (∼1.0 vs. 1.5 column per hour) [228, 4715], possibly accelerated by the higher titer of ecdysone [457]. Also, the proneural gene ac turns on anterior to the MF for the ﬁrst time [597]. Thus, the regions behind vs. ahead of the MF may be inﬂuenced differently by factors that control SOP initiation, but we do not yet know how.

The MF operates like a moving A/P boundary The MF of the eye disc resembles the A/P boundary of the wing and leg discs insofar as Hh activates dpp at the border when it diffuses anteriorly from the P region [457, 2466, 2632, 3238]. Despite this shared “Hh dpp” link, hh expression behind the MF is not under en control [572, 4168], so the MF cannot be moving by a “Hh en hh” loop (cf. the Minotaur Scenario, Ch. 5). The obvious differences are that (1) the MF is not a compartment boundary [2933], and (2) the MF moves [571, 1784, 4387]. Thus, the MF is a state-altering machine wherein cells switch from one transient identity to another until they settle upon their ﬁnal fate (cf. the Cabaret Metaphor, Ch. 4). At ﬁrst glance, Hh seems irrelevant for MF movement because the MF can traverse and “assimilate” (i.e., create ommatidia in) tissue that is deaf to Hh (e.g., smo null clones [1077, 1616, 4169]; cf. hh null clones [1082, 2632, 4169]). Similarly, Dpp seems irrelevant because the MF can traverse and assimilate tissue that is deaf to Dpp (e.g., clones that are Mad null [4648], tkv null [570, 1616], or punt LOF [570]; cf. large dpp null clones [570, 1788, 4648]). According to the “MF-pushing Model I,” proposed in 1993 by Heberlein et al. [1788] and Ma et al. [2632], Hh is supposed to diffuse anteriorly to turn dpp on in the MF, with Dpp somehow reciprocally enabling hh to turn on in R cells. However, this “Hh dpp hh” loop is disproven by the ability of the MF to traverse singly deaf clones that should be disabling the loop. Nevertheless, hh must be involved because the MF arrests in t.s. hh LOF mutants when they are heated [2632]. Dpp was also thought to participate in MF movement because the MF at least slows down (if not stopping completely) when t.s. dpp LOF mutants are heated [724].

230

IMAGINAL DISCS

Key: bristle no bristle displaced bristle

D P

A V

Rule: Every has a anterior to it. FIGURE 7.8. “Fickle Bristle Mystery” of the ﬂy’s eye. The entire left eye of a wild-type female specimen is schematized (cf. key).

Bristles (black symbols) occupy vertices in the lattice of facets (hexagons), but their pattern is less perfect. Directions (compass) are A (anterior), P (posterior), D (dorsal), V (ventral). The general rule is: “Bristles reside at the A end of each horizontal interface between ommatidia.” Bristles that obey this rule are indicated by solid black circles. Wherever this rule is obeyed, eye bristles occupy alternating vertices around each facet, with tertiary pigment cells at the remaining vertices (cf. Fig. 7.2). The rule is violated at the A and P margins, where bristles are missing (unﬁlled circles). It is also broken by bristles that lie at P ends of horizontal interfaces (triangles). These sporadic “errors” occur most frequently near the A margin. They exhibit their own sort of rule (lower right): “Wherever a bristle arises at the P end of an interface, none forms at the A end.” This correlation implies that (1) one bristle SOP arises per interface, and (2) this SOP is free to move to one end or the other [1804, 4715]. In other words, the errors are actually displacements (cf. Fickle Sensilla Mystery; Fig. 5.12g). Redrawn from [3539]. N.B.: The ﬂy eye is nearly circular: there are 32–34 columns in a typical wild-type female, with ∼32 ommatidia in the tallest column [2571, 3539, 4715]. The absence of bristles from the front of the eye is probably due to the remnant of wg-on tissue that is left after the D and V margins of wg-on cells have shrunk (as the MF encroaches anteriorly) [2554]. Wg’s role as a diffusible “bristle suppressor” is shown by (1) wg GOF clones which scar the eye and, in so doing, delete bristles from a swath of facets on either side of the scar [4390]; (2) sevE-wg eyes, which lack all bristles due to expression of wg in every facet under the control of the sev enhancer [518, 597, 3659]; and (3) naked cuticle LOF eyes whose bristleless margin widens due to an increased cellular responsiveness to Wg [3659]. We do not know why bristles are normally absent from the rear of the eye. In other insects, the interommatidial bristles obey similar rules but vary in density [1853]. The number of bristles per vertex increases in E(Elp)24D LOF eyes [4034].

CHAPTER SEVEN. THE EYE DISC

Also, dpp LOF ﬂies have small or missing eyes [2739, 4033], although this trait has been traced to a separate role for Dpp in MF initiation (see below). The aforementioned mosaics do offer one useful clue to solving this mystery: the MF slows down within the deaf patch, and the slowing is more pronounced at the center of the patch. This nonautonomy implies local rescue by an agent “X” that diffuses in from the surrounding wild-type tissue. Because the rescue is seen regardless of whether the patches are deaf to Hh [1616, 4169] or Dpp [570, 1616], X might be able to rescue either kind of tissue. Alternatively, Hh and Dpp might be redundantly serving as the rescuer for one another [1780]. Indeed, when cells are made simultaneously deaf for both Hh and Dpp (smo null Mad null clones [930] or smo null tkv null clones [1616]), they fail to support either MF movement or photoreceptor development. Evidently, therefore, the basic logic of MF progression is {Hh or Dpp}

MF movement.

The notion that Hh and Dpp act redundantly is counterintuitive for several reasons. First, Hh and Dpp do not affect one another reciprocally: Hh activates dpp (albeit in only part of the anterior region) [457, 2466, 2632, 3238], but there is no evidence that Dpp activates hh. Second, Hh and Dpp are distributed quite differently relative to the MF (Fig. 7.9). If Hh and Dpp are truly redundant, then they should behave similarly with respect to initiating extra MFs when they are ectopically expressed. In fact, however, they do not. Extra MFs (anterior to the endogenous MF) can be induced by goading either the Hh or Dpp pathway, but only the MFs induced by Hh-pathway stimulation then go on to express atonal and make photoreceptors. The full inventory of manipulations that can spark new MFs is listed below: 1. Locally activating the Hh pathway via hh GOF clones [722, 1077, 1786], ptc LOF clones [722, 723, 2631, 3562, 4167, 4572], or DC0 LOF clones [1082, 1788, 3238, 4167, 4171, 4572]. 2. Locally activating the Dpp pathway via dpp GOF clones [724, 3388] or by overexpressing dpp using dppGal4:UAS-dpp [724] or other Gal4 drivers [3388]. 3. Locally blocking Wg transduction via arr LOF clones [4573] or ubiquitously blocking Wg signaling by exposing t.s. wg LOF mutants to high temperature [2631, 3562, 4390]. Conceivably, dpp GOF (unlike hh GOF ) is acting indirectly by suppressing wg and thereby enabling Hh to

231

launch a MF from the dorsal anterior margin [1788] (i.e., “Dpp Wg Hh MF initiation” vs. “Dpp MF initiation”). The argument for an indirect role is bolstered by the aforementioned fact that tkv Q253D (activated receptor) clones fail to undergo neuronal differentiation, in contrast to hh GOF clones, which do [570, 1616, 1786]. It is also supported by several other ﬁndings: 1. Ectopic Dpp only induces extra MFs at the margins [3388] where hh is expressed in young discs [1082], implying a reliance on hh (although the “hot spot” for MFs at the A margin does not match Hh’s high point and may involve a “Dpp hh” link [406]). This reliance is proven by the inability of MFs to arise in discs where dpp is overexpressed ( via dpp-Gal4:UAS-dpp) sans hh function (due to hh LOF ) [406]. In the interior of the eye ﬁeld, dpp GOF clones behave like tkv Q253D clones: they fail to make R cells [3388], presumably due to the paucity of Hh there. 2. Extra MFs in wg LOF mutants also tend to arise at the margins [2631, 4390] – typically the D and A margins (but not the V margin where a redundant Wnt may be acting) [4387]. Again, the suspicion has been that removing Wg allows Hh to spark a new MF. This suspicion is conﬁrmed by the inability of wg LOF to induce new MFs when combined with hh LOF [406]. 3. Hh cannot be inducing MFs indirectly via Dpp (or Wg) because goading the Hh pathway via DC0 LOF induces MFs even when triply mutant (DC0 LOF dpp LOF wg LOF ) clones are blocked from emitting Dpp [1082]. Nor do DC0 LOF cells need to “hear themselves talk” because MFs are induced by DC0 LOF smo LOF clones [4169]. In 1999, Greenwood and Struhl stated the paradox clearly [1616]: how can Dpp rescue photoreceptor differentiation within smo null clones if Dpp cannot evoke such differentiation ectopically? To solve it, they proposed a “MF-pushing Model II,” which again invokes a diffusible factor X (cf. a similar model by Strutt and Mlodzik [4169]): 1. Hh {Dpp and X}. 2. {Hh or X} atonal photoreceptor differentiation and MF movement. Their data suggests that X may act through dRaf via a RTK receptor other than Egfr. However, that receptor should still be present in smo null tkv null cells, so X should be able to rescue them in a paracrine manner. Because it does not, the model must be ﬂawed. The ﬂaw can be easily ﬁxed though by supposing that X only acts in

232

IMAGINAL DISCS

b

D

MF

V

a

R1

m BarH1

R2

outer

P

A

rough

glass

R3

or

R4

svp lozenge

dPax2

cut

R5

pros spalt ?

MB

R8

MB

he ls

3 IG

ith

el

ia

c

R7

et

MF

inner

R6

ep

4

3 2

2 8 5

1 8 6 MB

MF

cell

apical

7

4 5

basal

d asynchrony

G1

Egfr OFF Egfr ON

EARLY LATE

synchrony

S G2 M

e

Ci-155

e

Gene expression patterns CycA,B CycE String CycD emc hairy Hh Ci-155 dpp da

sca

atonal

e sca

Rl-act m rough glass lozenge

IC

IG IG IG

... ... ... ...

...

asynchrony

CHAPTER SEVEN. THE EYE DISC

233

conjunction with Dpp: 1. Hh {Dpp and X}. 2. {Hh or [Dpp and X]}

Step 1. Step 2. atonal

etc.

Now, all the key facts can be explained: 1. A smo null clone is rescued because Dpp and X can both diffuse in. 2. A tkv null clone is rescued because Hh can diffuse in. 3. A smo null tkv null clone is not rescued because X alone is impotent. 4. MFs sparked by ectopic Hh can make ommatidia because they have Dpp and X. 5. MFs sparked by ectopic Dpp cannot make ommatidia because they lack X. According to this reasoning, the cycle of events in the MF “engine” would be as follows:

Step 3. Step 4. Step 5.

Hh is produced by R cells behind the MF. Hh diffuses anteriorly to induce Dpp, X, and atonal. Dpp and X jointly activate atonal. Atonal enables cells to adopt the R8 fate. A new column of R8p cells is created, thus rekindling the process.

Other riddles remain, however. For instance, expressing Hh ubiquitously should induce MFs everywhere, but only one new MF appears when hs-hh larvae are exposed to high temperature [2466]. This extra MF lies parallel to the endogenous MF and ∼1 MF-width ahead of it. Apparently, the “Hh dpp” link operates only in a certain part of the prospective eye region [1788, 3388] (cf. its restrictions in the leg disc, Ch. 5). Another odd ﬁnding is that overexpressing Hh in R8p cells (via

FIGURE 7.9. Genetic logic of eye development and dorsal-ventral stripes of gene expression in the eye disc epithelium during the 3rd-instar and early pupal period. See also Appendix 7. a. Mature left eye disc. Vertical stripes are the morphogenetic furrow (MF) and mitotic band (MB). Compass (above) gives axes in the fate map (cf. Fig. 7.1). The MF traverses the epithelium from P to A, leaving a hexagonal lattice of nascent ommatidia (dots) in its wake. The MB is needed to supply enough cells to ﬁnish assembling the ommatidia [1003, 3655, 4712]. b. Circuitry among genes that encode transcription factors for photoreceptor cell fates ( activation; inhibition; see text). Abbreviations: pros (prospero), svp (seven-up). BarH1 is one of two genes (the other being BarH2) in the Bar Complex. A combinatorial code for R cells is thought to exist [1272], although there is little evidence for it except for rough and svp, which dictate outer-type identity, and even there the data is murky. Neither rough nor svp can convert R7p to an outer state in a sev null background [232, 1856], so R7p must require the Boss-Sev reaction to become neural [4551]. Also, Rough and Svp are not interchangeable: they have different effects when expressed in cone cells [264, 4551]. c. Imaginary slice of the columnar epithelium (magniﬁed from box in a) at one instant of time. The MF is a groove (black) that runs along the D-V axis. It moves from P to A (long arrow). Each nascent ommatidium that it creates undergoes a series of transformations (short arrows). These stages can be seen in the (static) A-to-P sequence of nodes along each row because each successive column is older. The “intermediate group” stage (IG) precedes the rosette stage (not shown; cf. Fig. 7.5) [2042]. Within each IG, 2–3 R8p-like cells express senseless (not shown) [1279], which then turns off in all but one cell that becomes R8. Ovals (top view) are apical proﬁles of single cells (mystery cells not shown). Vertical lines (side view) are cell boundaries. The epithelium is actually thicker, and cell packing is less regular [4715]. d. Stages of the cell cycle. The span of each stage along the A-P axis is indicated by a black bar. As cells enter the furrow, they synchronize but lose this synchrony around the time that they begin mitosis [4715]. Arrows mean that a cell’s EGFR pathway must be off for it to go from G1 to S but must be on for it to go from G2 to M [185]. String is the downstream gating factor for the latter transition [185]. e. Domains of gene or protein expression relative to eye epithelium in c. Each stripe of gene expression along the D-V axis (parallel to the MF) is shown here in terms of its width along the A-P axis. Degrees of gene expression are indicated by shades of gray or by graded slopes. If LOF effects have been found, then the name of the gene/protein is in white letters on a black rectangle. Error bars indicate conﬁdence limits. Black ovals denote expression in IGs or earlier “initial clusters” (ICs) [4208], whereas a black frame (hollow oval) denotes expression between IGs. Thus, sca exhibits a punctate pattern of spaced islands (IGs), while atonal displays a wider stripe with intense A (IC) and P (IG) foci and a P edge that describes a sine wave [183, 184, 1921, 2461]. ICs and IGs are out of phase relative to one another (not shown), and the IC phase is dispensable [1616]. Genes or proteins: atonal [1077, 2493] (autoregulation [4208] ; links to glass [2042], hairy [2042] , and sca [1077]), Ci-155 (the activator form of Cubitus Interruptus) [1077, 1616] , CycA–E (Cyclins A–E) [185] , da (daughterless) [506] , dpp (decapentaplegic)[725, 3380], emc (extramacrochaetae) [508], glass [2569] , hairy [2540] , Hh (Hedgehog) [287] (autoregulation [1786, 3238]), lozenge [639] , mδ [2549] , Rl-act (activated Rolled; a.k.a. MAP kinase [332]) [2493] , rough [725] , sca (scabrous) [183] , and String [185] . Wiring at left ( activation; inhibition; circled “e” = extracellular signal) shows some genetic interactions (see text or references above). Links in panel b are discussed in the text (cf. Table 7.1). Panel c is adapted from [4715] and d is based on [185, 4291, 4293]. Panel e is styled after [1786, 1788, 4387], with additional data from the above references. For further circuit analysis, see [395, 4387]; for circuits that affect nonoptic head structures, see [80, 3380, 3663--3665, 4654]; and for comparable analyses in other systems, see [309, 2093, 4289].

234

IMAGINAL DISCS

109-68-Gal4:UAS-hh) does not perturb eye development, nor does overexpressing Hh in all cells behind the MF (via GMR-Gal4:UAS-hh) [4621].

Dpp and Wg control the rate of MF progress Another observation made by Greenwood and Struhl indicates a role for Dpp in pacing the furrow: tkv Q253D (activated receptor) clones cause the MF to move faster. This acceleration is attributable to a “Dpp hairy” link uncovered in the same study. Hairy is one of two antineural regulators of atonal that are expressed just ahead of the MF [508] (cf. Fig. 3.12). The other is extramacrochaetae: “{Hairy or Emc} atonal”. The stripe of emc-on cells precedes the hairy-on stripe, which in turn precedes the dpp-on stripe (Fig. 7.9). Although h LOF and emc LOF exhibit negligible effects in the adult eye, patches of doubly mutant h LOF emc LOF tissue allow the MF to outpace ﬂanking wild-type tissue by as many as 8 columns [508]. Because Dpp is under Hh control, Hh has opposing effects on atonal at different distances [1784, 1786, 4387]: Short-range activation: Long-range inhibition:

{Hh or [Dpp and X]} atonal. dpp hairy Hh atonal.

In contrast to its role as a brake far from the margins, Dpp acts an accelerator along the D and V margins, and Wg becomes the brake [2631, 3562, 4390]. (Wg also retards the MF per se [4390].) At each tip of the MF, the dpp-on stripe extends a perpendicular arm anteriorly into the wg-on patch (Fig. 7.3). The arms must be under separate genetic control because (1) they persist when hh LOF causes the main dpp-on stripe to disappear [406, 2632], (2) they are less sensitive to suppression by rough GOF [4389], (3) they are the ﬁrst dpp-on domain to disappear when dpp expression is artiﬁcially reduced [724], and (4) they are the only dpp-on cells to exhibit autostimulation (Dpp dpp) [724]. The latter feedback loop pushes the dpp-on domain into wg-on territory independently of the MF engine described above: Step 1. Step 2.

Dpp spreads anteriorly from the “arm” cells into the wg-on region. Once there, Dpp turns off wg and turns on dpp, thus extending the arm.

Given the nature of the feedback loop, Dpp diffusion should drive dpp expression into the interior of the eye ﬁeld as well [724, 3388] (cf. the Minotaur Scenario, Ch. 5).

Because it does not, some unknown agent must keep the loop at bay. The evidence for a mutual antagonism between Dpp and Wg in the arm regions – and elsewhere in the eye ﬁeld – is as follows: 1. Overexpressing Dpp around the perimeter of the eye (via dpp-Gal4:UAS-dpp) abolishes the V patch of wg-on cells and reduces the D patch to a tiny remnant in the ocellar region [724]. The same effect is seen with randomly induced dpp GOF clones [3388]. Implication: “Dpp wg”. 2. Loss of Dpp from the P margin of the eye (in dpp LOF mutants [724, 4648] or Mad LOF clones [4648]) is associated with de novo expression of Wg there. Implication: “Dpp wg”. 3. When wg is suppressed by heat-treating t.s. wg LOF mutants, the posterior arc of dpp-on cells expands anteriorly to occupy what used to be the wg-on areas along the D and V margins [2631, 4390]. Implication: “Wg dpp”. 4. Randomly induced dpp GOF clones can stimulate the endogenous dpp gene in marginal regions, except where wg is expressed [3388]. Implication: “Wg dpp”. Despite the similarity of this antagonism to the circuitry of leg development (cf. Ch. 5), Wg’s effect on Dpp appears to be at a post-transcriptional level [1780, 4390]. Moreover, dpp is here playing only a permissive (vs. instructive) role: it coordinates the rates of movement of the tips with the MF proper [4387]. Dpp inﬂuences the MF rate by modulating the cell cycle [3330], speciﬁcally by promoting G1 arrest [1903]. Indeed, the famous groove that forms the MF may be an irrelevant side effect of mitotic synchrony. In the wing disc, two MF-like furrows ﬂank the D/V line apically [498, 500, 1178] at zones of mitotic quiescence [2079, 2846, 3374, 3979, 4683] while a single groove runs basally, and an ectopic furrow has been artiﬁcially created along the wing’s A/P border by using ptc-Gal4 to drive a dominant-negative dCdc42 allele [1133].

The MF originates via different circuitry The MF arises where the D/V (mirr-on/off) interface intersects the P margin [696]. This intersection point normally coincides with the optic stalk, but the stalk per se cannot be causal because the MF can be uncoupled from it. To wit, when the mirr-on/off line is pushed ventrally relative to the stalk (due to dpp-Gal4:UAS-wg [1781] or ey-Gal4:UAS-pannier [2752]), the MF starts at the

CHAPTER SEVEN. THE EYE DISC

ventral site where this line touches the margin. The circuitry of MF initiation differs from the circuitry of MF progression in other features as well [1784, 4387]. During initiation, atonal is activated in four spaced clusters of cells just anterior to a hh-on zone at the rear of the disc [1077, 1082]. The posterior two are unusual insofar as they never acquire all 8 R cells (one stops at 3, the other at 5), and both of them disappear without being incorporated into the ﬁnal lattice [4364]. Thus, the next column (5 clusters) comes to occupy the rear edge of the ﬁnal array [4364]. Nevertheless, the MF can arise (and move slightly) without either atonal expression or photoreceptor maturation [2042]. This ability is a salient difference from MF progression, which does require R cells [1786, 1788]. Other odd aspects of the eye’s P edge include 1. The P margin is the only part of the eye where the Notch pathway is not required for proneural competence [183]. 2. The EGFR pathway is required there for MF initiation [1083, 2355], whereas it is dispensable elsewhere for MF movement [2355]. Before MF initiation, dpp is expressed at the D and V margins and weakly at the P margin (cf. Fig. 7.3) [930, 2739]. At this same time, four “early eye” genes are also expressed [930] (HB = homeobox): eyeless (ey, HB), eyes absent (eya), sine oculis (so, HB), and dachshund (dac). Three of the latter genes are expressed in the P half of the eye region with an intensity that fades with distance from the P margin [397, 759, 930, 2689]: eya, so, and dac. The fourth (ey) is expressed throughout the eye region [930, 1684, 3486]. Four other HB genes – eye gone (eyg) [1780], twin of eyegone (toe) [2022], optix [3851], and twin of eyeless [933] – may also belong to this “early eye” group. MF initiation requires Dpp signaling [723, 724]: whenever a clone that is deaf to Dpp (e.g., Mad LOF [1780, 4648], punt LOF [570, 1780], or tkv null [570]) resides at the P margin, the MF fails to launch at that site. MF initiation also requires the early eye genes because no functional MF forms when any of them is disabled [1684, 2042, 2689, 3387]. What is the circuitry? Dpp evidently acts through eya, so, and dac because their expression vanishes in Mad null clones that touch the margin [930], although expression of ey persists. Dpp {eya, so, and dac} ey MF initiation.

MF initiation.

The “Dpp {eya, so, and dac}” link does not operate during MF movement because internal Mad null clones

235

express eya, so, and dac [930]. (Inter se links among the early eye genes are considered in Ch. 8.) MF initiation also requires hh signaling (despite prior evidence to the contrary [1788, 2632]): no MF originates when t.s. hh LOF mutants are heated during the initiation stage [406] (cf. hh null clones [1082]). Hh is normally detectable along the P edge in late-2nd/early-3rd instar [406, 1082, 3665] (despite a report to the contrary [2632]). Within this zone, hh may act upstream of dpp because dpp expression there vanishes when young t.s. hh LOF larvae are heated [406, 3665], although ptc LOF clones at the P margin can rescue aborted MFs in dpp LOF eyes [723]. In turn, the Iro-C must act upstream of hh because erasure of the Iro-C on/off border (by ubiquitous expression of ara via ey-Gal4:UAS-ara) prevents Hh levels from rising high enough to spark a MF [696]. Finally, LOF-GOF analysis shows that (1) pannier (pnr) regulates the Iro-C via Wg [696, 1781, 2752] (cf. the notum [1380]) and (2) the Iro-C on/off border controls hh via the Notch pathway [696]. Thus, the basic chain appears to be Wg Iro-C on/off Notch Pnr {eya, so, and dac} MF initiation.

Hh

Dpp

During the latter half of the 2nd instar, Notch is needed throughout the eye part of the disc (not just at the D/V border) to stop it from developing as an antenna (cf. Fig. 6.9) [2353]. At this time, the EGFR pathway acts antagonistically to Notch [2353]. Both pathways probably affect the above chain at the {eya, so, and dac} node (cf. Fig. 7.3 and [1189]). Indeed, Eya cannot function without being phosphorylated by dMAPK [969]. When ectopic MFs are elicited anterior to the endogenous MF, they spread radially from the inception point like a ripple in a pond [723, 2036, 2631]. This independence from the global framework of the disc argues that the MF is a self-propagating wave. Evidently, the P-toA motion that characterizes insects is arbitrary: evolution could have launched the MF from any point in the prospective eye ﬁeld. Indeed, the only reason that MFs do not arise at the D and V margins is Wg’s damping effects there [724, 1781, 2631, 3562, 4390]. The eye ﬁeld thus constitutes the sort of excitable medium that has been simulated by a fascinating genre of models known as “cellular automata” [824, 1661, 4341]. Cellular automata are imaginary grids of “cells” that behave like parallel computers. Each cell adopts a state based on inputs from its neighbors, and its new state then inﬂuences their states in the next round of computation (cf. John Conway’s popular game of “Life” [74, 1381, 1382]). A variety of dynamic patterns can be created

236

by changing (1) the interactive rules and (2) the initial conﬁguration of states [32, 1437, 1769, 2694, 4720]. These systems nicely illustrate how local rules can generate global patterns as emergent properties. No simulations of eye circuitry per se have been attempted since the Hh-DppWg rules were ﬁgured out, but they may be a useful way of testing models in the future [4005]. When an extra MF moves in an A-to-P direction, the ommatidia that it creates have a reversed A-P polarity

IMAGINAL DISCS

[722, 4572].

D-V polarity, however, tends to be normal, as expected given the integrity of the Iro-C circuitry [3562, 3563]. Exceptions include (1) interior wg GOF clones [4390] that activate mirr [1781] and (2) marginal clones that may affect the early phase of equator initiation [722, 723, 1781, 4167, 4648]. (See [2631, 4573] for partial theories and [2752] for overview.) When opposing MFs collide, they typically merge to form an amazingly seamless ommatidial array [724, 3388].

CHAPTER EIGHT

Homeosis

Few phenomena are as entrancing as the transformation of one thing into another. The ancients believed that sorcerers had such powers, and modern magicians can still fool children with illusions of this sort. A special class of mutations can actually accomplish this feat. “Homeosis” means a transformation of one body part into another [3214, 4486]. The term was coined by William Bateson to describe deformities that are occasionally found in nature. In his classic 1894 monograph, Bateson cataloged 886 abnormal biological specimens [240], many of which exhibited homeosis. His intent was to investigate how anatomy varies as a way of comprehending how evolution works [2509]. This goal was obvious from the book’s overtly Darwinian title: “Materials for the Study of Variation Treated with Especial Regard to Discontinuity in the Origin of Species.” Variation has been supposed to be always continuous and to proceed by minute steps because changes of this kind are so common in variation. Hence it has been inferred that the mode of variation thus commonly observed is universal. That this inference is a wrong one, the facts will show. . . . The evidence of discontinuous variation suggests that organisms may vary abruptly from the definite form of the type to a form of variety which has also in some measure the character of definiteness. Is it not then possible that the discontinuity of species may be a consequence and expression of the discontinuity of variation?. . . For the word ‘metamorphy’ I therefore propose to substitute the term homœosis, which is also more correct; for the essential phenomenon is not that there has merely been a change, but that something has been changed into the likeness of something else. [240]

Ever since Bateson, the “Homeosis Riddle” has been: how do mutations cause such phenotypes? The ability

of single mutations to drastically alter the anatomy led to speculation that species might have arisen saltationally via such “macromutations” [1177, 3619]. This idea was dramatized in Richard Goldschmidt’s “Hopeful Monster Hypothesis” [1056, 1521, 1523]. (See [551, 1582, 1584, 4113, 4529] for critiques.) That hypothesis has experienced a revival [662, 4258, 4520] in the wake of Halder et al.’s 1995 ﬁnding that eyes can be induced nearly anywhere on the ﬂy surface by misexpressing the gene eyeless (see below) [1685]. In the Fly World, the mystique of homeosis has been compounded by transdetermination (cf. Ch. 4) [1670] and by the ability of physical or chemical agents to “phenocopy” many of these monstrosities in wild-type ﬂies [1522, 1524, 1667, 2398, 2399]. The weirdest example of phenotypic mimicry is the bithorax phenocopy: wild-type ﬂies can acquire a UbxbxLOF phenotype (notum and winglets instead of halteres) if they are exposed to heat shocks [1826, 2636] or ether vapor [1511] as blastoderm stage embryos [427, 646, 1859]. The implication is that Ubx expression is transiently unstable at the front of the 3rd thoracic segment [647, 1469, 1860, 4258], but teratogenic effects there are more complicated [3751, 4595], and it is still unclear why other homeotic genes are not similarly affected elsewhere in the embryo.

BX-C and ANT-C specify gross metameric identities along the body In Drosophila there are two major clusters of homeotic genes (i.e., genes that cause homeosis when mutated) [2421, 2561, 2656]. Both reside on the right arm of the 3rd chromosome, and together they control the identities of all embryonic parasegments (PS) [162, 2166, 2934]. 237

238

Expression:

a

-120 kb

PS 5-13

-80

PS 6-13

-40

Ubx

-30

PS 8-13

PS 12-13

40

80

120

2 recording

Ubx

b

0

PS 7-13

160

abd-A

-20 kb

-10

PS 13

200

Abd-B

3 emory

0

10

m

(bxd)

ac

va t or i o n re ssio n

ti

1

(pbx)

PRE

s s sc iscs iscs isc i d ll d ld d D a al V imaginal enhancers

re

p

embryonic PS enhancers (includes pbx)

+

+

Ftz

Twist

footprints

c

d Kni ?

En ?

Hb

Tll

TTTGGTTTTTTACCAACAGCCTTTGGAA

Kr ?

CHAPTER EIGHT. HOMEOSIS

1. The Bithorax Complex (BX-C, 315 kb) has 3 homeotic genes (Fig. 8.1), which dictate fates in the abdomen and posterior thorax (PS 5–14) [2703] : Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B). 2. The Antennapedia Complex (ANT-C, ∼330 kb) includes 5 homeotic genes that dictate fates in the head and anterior thorax (PS 1–5) [1023, 2167] : Antennapedia (Antp), Deformed (Dfd), labial (lab), proboscipedia (pb), and Sex combs reduced (Scr). The ANT-C also has other types of genes [1025, 2717, 4671], including the segmentation genes bicoid and ftz, which seem to have acquired new functions during dipteran evolution [1467, 2590, 4067]. Until 1984, when the “homeo” box was revealed, researchers could only speculate about the nature of homeotic genes [3882]. The homeobox is a ∼180 b.p. sequence that encodes a DNA-binding domain (cf. App. 1)

239

[1417, 2498].

It is shared by genes in both the BX-C and ANT-C [2783, 2785, 3844]. Not all homeotic genes have a homeobox (Table 8.1) [1418], but the motif’s widespread conservation within this group implies that evolution enlisted the homeobox family for administrative roles [45, 46, 1412, 1414, 4153] that were not bestowed on other families of transcription factors in animals (e.g., zinc ﬁnger) [3837], although the MADS box serves an analogous function in plants (cf. App. 1) [3105, 3477]. Most of the ﬂy’s homeotic genes fall into 3 functional classes [662]): 1. “Metamere identity” genes of the BX-C and ANT-C establish segmental or parasegmental identities (or groups thereof ) along the body column [162, 1429]. The term “Hox” (contraction of homeobox) was coined to designate homologous complexes in vertebrates [48, 3677, 3839] (cf. reviews [658, 1223, 1236, 3871]), but it is also used more broadly to include the two clusters in ﬂies [1429, 1591, 2778, 2937, 4563]. As explained below, two other

FIGURE 8.1. The Bithorax Complex (BX-C) and its regulation during development. See also Appendix 7.

a. The BX-C contains 3 protein-encoding homeotic genes: Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) [3740]. Exons are drawn as black rectangles, with introns indicated as thin (kinked) connecting lines. When reporter genes (e.g., lacZ ) insert into this 320-kb region, their expression is conﬁned to a speciﬁc span of parasegments (PS, above) [284, 2773] due to nearby enhancers in the BX-C (cf. Fig. 4.2 for PS numbering) [2987, 3480, 3481, 3940, 4864]. The more rightward the reporter’s insertion point (= distal on the right arm of the 3rd chromosome), the more the expression domain recedes toward PS13. This colinearity of enhancers (proximal to distal) relative to body regions (anterior to posterior) is also seen in the order of “iab” genetic control elements (not shown) [416, 699, 907, 3738]. The reason for the colinearity is unknown [162, 686, 3511, 4152, 4328, 4498], although clever guesses have been offered [1113, 1400, 2191, 2295, 2508, 2672, 3494]. A similar “Homeobox Homunculus Mystery” [1417, 1805] exists for Hox gene complexes of vertebrates [1111, 1112, 2266, 2292, 4446] – i.e., why are Hox genes and enhancers arranged like the parts of a miniature man or ﬂy? b. Enlarged view of the 40-kb section upstream of Ubx. Three stages in regulation are depicted. In Step 1, embryonic enhancers (shaded rectangles) turn Ubx on or off in speciﬁc parasegments based on input that they receive from segmentation genes (c; cf. Fig. 4.2). In Step 2, the PRE (Polycomb Response Element, black oval) site indelibly “records” the cell’s state by recruiting activator (Trithorax-Group) or repressor (Polycomb-Group) proteins [3196], depending on whether Ubx is actively transcribed (proteins not shown). Finally, in Step 3, the recruited proteins affect the imaginal disc enhancers (black rectangles) by blocking or aiding nucleosomal silencing of the DNA within ≥10 kb. Among the four disc enhancers, two drive expression in all discs, one affects mainly ventral (leg) discs, and the other affects mainly dorsal (wing and haltere) discs. c. Magniﬁed view of the pbx embryonic enhancer (619 b.p.), showing footprints (bars) of various regulatory proteins. ¨ Abbreviations: En (Engrailed), Ftz (Fushi tarazu), Hb (Hunchback), Kni (Knirps), Kr (Kruppel), Tll (Tailless). All these proteins participate in ectodermal segmentation (cf. Fig. 4.2 and [3680] for tailless, a terminal gap gene) except Twist, a bHLH transcription factor that activates mesodermal target genes [249, 1985, 2842, 4818] in the embryo’s D-V patterning system [3576, 4290]. Plus signs indicate activation and minus signs denote repression, although individual footprint sites have not been tested for function in vivo [3397]. Kr and Kni can repress BX-C genes [680, 3902], but expression of a pbx-driven lacZ reporter is normal in Kr LOF or kni LOF embryos [4864], arguing against pbx-mediated regulation of Ubx by these agents. En is a repressor for Ubx in the haltere disc [1138, 1162, 1635, 4229], but in the embryo it can behave as either an activator [3481] or a repressor [684, 2670, 2725]. d. Nucleotide sequence in an enlarged section the pbx cis-enhancer, where a Twist binding site (bar above) appears to overlap a Hb binding site (bar below). It is important to remember that such sites are delimited by DNase protection assays, so the actual contact area with DNA may be smaller. Hence, these proteins might be able to bind simultaneously (i.e., without competing). Demarcations of PS expression zones in a are only crude estimates due to a limited number of insertion sites [284]. Exons for BX-C genes are after [284, 2773, 3156, 3902], although each of the three genes has other splicing isoforms. Nevertheless, for Ubx at least, the isoforms act alike with regard to specifying parasegment identity [684]. Map coordinates obey convention [283]. Schematic in b is adapted from [3397]. See [3667, 4319] for ﬁner dissections that identify separate Pc-G and Trx-G binding sites (PRE vs. TRE). Data in c and d are from [3397, 4864].

240 TABLE 8.1. GENES THAT CAN HOMEOTICALLY TRANSFORM LEG, WING, OR EYE DISC DERIVATIVES INTER SE ∗ FROM:

TO:

Leg (L1, L2, L3)

Wing (W) or Notum (N)

Eye (E), Antenna (A), Head Capsule (H), or Palpus (P)

Leg (L1, L2, L3)

L1 L2: Scr-LOF (a.k.a. Msc) [3286] . Trx-G-LOF : See below**. {L2 or L3} L1: Pc-G-LOF : See below**. Ubx-LOF (Scr-on) [3311] . Antp-GOF (a.k.a. Scx) [2190] . Scr-GOF (a.k.a. Msc) [3285, 3286] . L2 L3: Pc-G-LOF : See below**. {Pc-LOF and abd-A-LOF and Abd-B-LOF} [582]. Ubx-GOF [1120, 2506]. L3 L2: Ubx-LOF [4627] . Trx-G-LOF : See below**.

L1 (dor.) W: wg-GOF (vg-on) [2082, 2753, 2754]. L2 (dor.) W: su(f)-LOF [3442]. vg-GOF [2219, 2252, 4564], whereas L3 makes haltere tissue [4564]. wg-GOF (vg-on) [2082, 2753, 2754]. {vg-GOF and wg-GOF} [2252]. {wg-GOF and Ser-GOF} (vg-on) [885, 2092]. L3 (dor.) W: wg-GOF (vg-on) [2082, 2753, 2754].

L (dor.) E: GOF in “early eye” genes: See below**. L (prox.) A: Dll-GOF (hth-on and spalt-on) [1085]. L (dist.) A: pb-GOF [413]. ss-GOF [1119]. {L1 or L2} A: {Antp-LOF and Scr-LOF} [2641, 4149]. {L1 or L2 or L3} A: {Antp-LOF and Scr-LOF and Ubx-LOF} [4149]. {Dll-GOF and hth-GOF} [1085]. L2 A: Antp-LOF [677] . {L2 or L3} A: {Antp-LOF andUbx-LOF} [4149]. L P: pb-GOF [93].

Wing (W) or Notum (N)

W L: dLim1-GOF [1218]. W (pouch) L: Dll-GOF (hth-on and spalt-on) [1085, 1561].

W N: LOF in Wg pathway: See below**. LOF (misc.): N-LOF [886], sd-LOF [538, 2015], tet-LOF [2015], or vg-LOF [2570] . GOF (misc.): Egfr-GOF [4543], ara-GOF [4543], or pnr-GOF (vg-off) [614]. N (pos.) W: LOF in EGFR pathway: See below**. hth-LOF (vg-on) [678]. GOF (misc.): dpp-GOF [2954], sd-GOF (vg-on) [4463], {vg-GOF and wg-GOF} [2252], {wg-GOF and Dl-GOF} (vg-on) [2252], or {wg-GOF and Ser-GOF} (vg-on) [885, 2252]. N (unsp.) W: GOF in Wg pathway: See below**. {vg-GOF and sd-GOF} [1686].

W (hinge) E: GOF in “early eye” genes: See below**. W (prox.) A: Dll-GOF (hth-on and spalt-on) [1085].

241 Eye (E), Antenna (A), Head Capsule (H), or Palpus (P)

A L1: Pc-G-LOF: sxc-LOF [1965]. Scr-GOF (ss-off [1119]) [4806] . A L2: Dll-LOF (ss-off) [1085, 3242] . exd-LOF [1543, 3527]. hth-LOF [3380] or hth-DN (minus HD) [3716]. pb-DN (altered HD) [3333]. Pc-G-LOF: See below**. ss-LOF [1166] (cf. heat sensitivity [1623, 3810]). tgo-LOF [1166]. Trx-G-LOF: See below**. GOF in Hox genes: See below**. Arp-GOF [513]. cut-GOF [2080]. tsh-GOF [3240]. A L3: Ubx-GOF [2676]. H (ant.-ven. ) L2: exd-LOF [1543].

E W: opht (LOF?) [2561, 3210--3212]. {Antp-GOF and N-GOF} (vg-on) [2362]. Dl-GOF [2092]. Opt (GOF?) [2561, 3439, 3788]. {wg-GOF and Ser-GOF} (vg-on) [2092]. {E or A or H} W: vg-GOF [2219, 3291, 3936]. E N: trx-LOF [1966]. H (pos.-dor.) N: Dfd-LOF [2830]. exd-LOF [1543, 3527]. hth-LOF [3380]. lab-LOF [2829]. Antp-GOF [3802]. Scr-GOF [4872]. H (ven.) W: wg-GOF (vg-on) [2082].

E A: LOF in Notch pathway: See below**. ey-LOF (?) [3746]. GOF in EGFR pathway: See below**. ci-GOF [2353]. Dll-GOF (hth-on and spalt-on) [1085] . E H: LOF (misc.): eya-LOF [1780], Mad-LOF [4648], {Mad-LOF and DC0-LOF} [4648], sgg-LOF [1833], slimb-LOF [2856], or ttk-LOF (?) [4773]. GOF in Hh pathway?: See below**. hth-GOF [3380]. A E: GOF in “early eye” genes: See below**. tsh-GOF [3240]. A P: pb-GOF [413] . wg-GOF [2082]. H (dor.) E: LOF (misc.): {ara-LOF and caup-LOF} [3380], dsh-LOF [1833], ee-LOF (?) [190], Iro-C-null [696,697], pnr-null [2752], or tfd-LOF (?) [2562]. tsh-GOF [3240]. H (ven.) E: exd-LOF [1543]. hth-LOF [3226, 3380]. GOF in “early eye” genes: See below**. N-GOF [2362]. H (unsp.) E: {Egfr-LOF and gro-LOF} [3465]. GOF (misc.): ara-GOF (rare) [696], caup-GOF (rare) [696], or dpp-GOF [3388]. H A: LOF (misc.): {ara-LOF and caup-LOF} [3380], Iro-C-null [697], or tfd-LOF (?) [2562]. Dll-GOF (hth-on and spalt-on) [1085, 1561]. ss-GOF [1119]. H (ven.) H (dor.): ara-GOF or caup-GOF or mirr-GOF [697]. H (dor.) P: Iro-C-null [697]. P A: ss-GOF [1119], wg-GOF (pb-on) [2082]. (continued)

242 TABLE 8.1 (continued ) ∗ The genes listed above are “homeotic” insofar as they can cause speciﬁc body parts to develop like other body parts, but only a subset has a homeobox [1418]. Conversely, there exist homeobox

y) indicate transformations (from structure x to structure y), and letter codes for structures are given in row and column headings genes that do not cause homeosis [1467, 2934, 3836]. Arrows (x (e.g., N = notum). Within each category, LOF effects are listed ﬁrst (alphabetically) followed by GOF effects. “and” means “in combination with.” Genes that are known to be on or off in the transformed regions are listed in parentheses. Homeotic switches between A and P compartments of the same disc or between D and V compartments are omitted (cf. Chs. 5 and 6), except for the eye disc, where different histotypes are involved. Directions: ant. (anterior), dist. (distal), dor. (dorsal), pos. (posterior), prox. (proximal), ven. (ventral), unsp. (unspeciﬁed subregion). “Weak” and “strong” refer to penetrance or expressivity or both. Genes (excluding Hh, Dpp, and Wg pathways; cf. App. 6), with DNA-binding motifs (if any) in parentheses (HD, homeodomain; bHLH, basic helix-loop-helix; HMG, high mobility group; ZF, zinc ﬁnger): abd-A (abdominal-A; HD), Abd-B (Abdominal-B; HD), Antp (Antennapedia; HD), ara (araucan; HD), Arp (Aristapedoid), ash1 and 2 (absent, small, or homeotic discs 1 and 2), Asx (Additional sex combs), brm (brahma), caup (caupolican; HD), ci (cubitus interruptus, ZF), crm (cramped), cut (HD), dac (dachshund), Dl (Delta), Dll (Distal-less; HD), Dsp1 (Dorsal switch protein 1, HMG), ee (extra eye), Egfr (EGF receptor), esc (extra sex combs), exd (extradenticle; HD), ey (eyeless; paired/HD), eya (eyes absent), gro (groucho), hth (homothorax; HD), Iro-C (Iroquois Complex; HD), kis (kismet), lab (labial; HD), mirr (mirror; HD), moira, mxc (multi sex combs), N (Notch), opht (ophthalmoptera), Opt (Ophthalmoptera), optix (HD), pb (proboscipedia; HD), Pc-G (Polycomb Group), Pc (Polycomb), Pcl (Polycomblike), pco (polycombeotic), pho (pleiohomeotic), pnr (pannier), pnt (pointed, Ets), Psc (Posterior sex combs), Sce (Sex comb extra), Scm (Sex combs on midleg; ZF), Scr (Sex combs reduced; HD), sd (scalloped), Ser (Serrate), slimb, so (sine oculis; HD), ss (spineless; bHLH), su(f ) (suppressor of forked), sxc (super sex combs), tet (tetraltera), tfd (two-faced), tgo (tango; bHLH), toy (twin of eyeless; paired/HD, trx (trithorax; ZF? [4066]), Trx-G (Trithorax Group), tsh (teashirt, ZF), ttk (tramtrack, ZF), Ubx (Ultrabithorax; HD), vg (vestigial). “Iro-Cnull ” means null for ara, caup, and mirr. When alleles cannot be ascribed to LOF or GOF classes, they are labeled with original superscripts, except for DN (dominantnegative) alleles, which typically disable the endogenous gene and lead to a LOF effect. Some GOF effects may be misleading [50, 1418] due to promiscuity of DNA binding by HD proteins [1767, 1868, 2119] (cf. App. 1). The gene eye gone (Pax-6 family; not listed) causes extra eyes (locations not reported) when it is ectopically expressed [2090]. Some of the listed defects involve whole body segments or parasegments [1120, 2784]. For example, the following affect not only legs but also wing or haltere (T2 = mesothorax; T3 = metathorax): T2-to-T3 (Pc LOF [4327], Ubx GOF [591, 673, 1547, 2921, 4624]) and T3-to-T2 (Ubx LOF [591, 2925, 4627]). In contrast, “ﬁeld-speciﬁc” genes cause homeosis to a certain histotype virtually anywhere in the body when they are misexpressed [662]. By this deﬁnition, vg would be a master gene for wing identity [2219]. Other deformities defy classiﬁcation. For example, duplicated antennae are seen in mirr LOF discs (where the fng-on area spreads dorsally into the ocellar region), but there is no compensatory loss of eye tissue [4797]. There is no simple correspondence between genes and body parts, except for the dorsal head, which appears to rely mainly on the Wg pathway [2631] under the control of Pannier [2752]. For a comparable table of transdetermination frequencies, see [3883]. For further data, see Flybase and [2561, 3165, 3214, 3881]. **Pathway details: L2: Trx-GLOF : ash1 LOF [3884] , ash2 LOF [757] , brm LOF [4243], Dsp1 LOF [1013], kis LOF [964], moira LOF [475], or trx LOF [451] . L1 {L2 or L3} L1: Pc-GLOF [2400] : Asx LOF [3970], crm LOF [4793], esc LOF [1509] , mxc LOF [3721], Pc LOF [3285] , Pcl LOF [2595] , pco LOF [3370], pho LOF [1500], Psc LOF [2704] , Sce LOF [453], Scm LOF [405] , or LOF [1967] . sxc L2 L3: Pc-G LOF : esc LOF [1508], pho LOF [1500], or Pcl LOF [1122]. L2: Trx-GLOF : ash1 LOF [3884] , ash2 LOF [757] , or trx LOF [1966] . L3 E: GOF in “early eye” genes: dac GOF (ey-on) [3894], ey GOF (dac-on, eya-on, and so-on) [1684] , eya GOF [394], toy GOF (ey-on) [933], or {dac GOF and eya GOF } [743]. L (dor.) W N: LOF in Wg pathway: wg LOF [4543] , arm LOF [3317], dsh LOF [3310], porc LOF [3310], skinhead LOF [3371], or Notum GOF [1494]. W: LOF in EGFR pathway: Egfr LOF (via argos GOF ) [4543], or dRaf LOF [207]. N (pos. ) N (unsp.) W: GOF in Wg pathway: wg GOF [207] (but only rarely [2252] unless strongly expressed [3025]), arm GOF [3025], or osa LOF [849]. W (hinge) E: GOF in “early eye” genes: ey GOF (weak; dac-on and eya-on) [744] , {ey GOF and dpp GOF } (strong) [744], eya GOF [394], toy GOF (ey-on) [933], {dac and eya} [743]. L2: Pc-GLOF : Pc LOF (ss-off [1119]) [2517], or pho LOF [1500]. A A L2: Trx-GLOF : ash1 LOF [3884] , ash2 LOF [757] , lawc LOF [4892], or trx LOF [1966]. L2: GOF in Hox genes that turn off hth and hence block nuclear import of Exd [156, 3527, 4806]: abd-A GOF (ss-off [1119]) [4806] , Abd-B GOF [676] , Antp GOF (ss-off [1119]) [4241] , or Ubx GOF A (ss-off [1119]) [4806] . E A: LOF in Notch pathway [2353]: N DN , Dl DN , or Ser DN . A: GOF in EGFR pathway [2353]: Egfr GOF , spitz GOF , Ras1 GOF , dRaf GOF , or pnt GOF . E H: GOF in Hh pathway?: hh GOF [722] or ptc LOF [722]. E E: GOF in “early eye” genes: ey GOF (eya-on) [394] , eya GOF [398] , or optix GOF [3851] . A H (ven.) E: GOF in “early eye” genes: dac GOF (ey-on) [743, 3894] , ey GOF (dac-on) [3894] , eya GOF [743], optix GOF [3851] , {dac GOF and eya GOF } [743], or {eya GOF and so GOF } [3387].

CHAPTER EIGHT. HOMEOSIS

groups of genes (Pc-G and Trx-G) yield similar phenotypes because they propagate the states of Hox gene expression (on or off) after embryogenesis. 2. “Field-speciﬁc” genes enforce identities in regions of the body that are not segments or parasegments [2410, 2677, 3716]. Examples include the Iro-C genes, whose on/off states assign eye cells to D vs. V compartments (cf. Ch. 7) [696], and the engrailed-invected pair, whose on/off states assign thoracic cells to A vs. P compartments (cf. Chs. 4–6) [4229]. Whether these outlying complexes were founded by “escapees” from the primordial Hox array is unclear. (See [153, 933, 1843, 2012, 2022] for more homeobox complexes in the ﬂy genome, [567, 568, 2170] for homeobox phylogeny, and [490, 1236] for the issue of dispersal frequency.) 3. “Cell type” genes specify histotypes at the single-cell level [282, 853, 1069, 1901, 3033, 4630]. An example is the homeobox gene cut, which controls sensillar identity (cf. App. 4) [378]. The rest of this chapter explores how genes in the ﬁrst two classes function. As for how genes in the third class work, a case study (the bristle) has already been presented in Chapter 2.

Ubx enables T3 discs to develop differently from T2 discs The classic example of homeosis in Drosophila is the four-winged ﬂy with two thoraxes [1214, 3608], hence the name “bithorax.” This phenotype entails a conversion of T3 into T2 (i.e., 3rd into 2nd thoracic segment). Wildtype T3 discs (haltere and 3rd leg) express much more Ubx than T2 discs (wing and 2nd leg) [496, 782, 4625], and a maximal T3-to-T2 transformation is achieved when Ubx is mutationally shut off in T3 by disabling its abx, bx, and pbx enhancers [2510, 2564]. In that case, the haltere becomes a wing [2506, 2924], and the 3rd leg resembles a 2nd leg in its A compartment, although its P compartment adopts T1 identity [2198, 2925, 3311] due to (1) nulliﬁcation of an embryonic “Ubx Scr” link [162, 686, 1549, 1772, 3321], (2) consequent derepression of Scr [2564], and (3) enforcement of a T1 state by Scr [2190, 2561, 3285, 3286, 4345]. The four-wing trait is atavistic [664, 1429, 1466, 4550] because dipteran halteres evolved from paleopteran wings [664, 4750]. Ed Lewis cited this atavism in 1978 when he proposed his “Ratchet Model” for the evolution of metameric identity [2507]. In that model, the T2 segment represents a “ground state” that reﬂects the ancestral condition.

243

Flies almost certainly evolved from insects with four wings instead of two and insects are believed to have come from arthropod forms with many legs instead of six. During the evolution of the fly, two major groups of genes must have evolved: ‘‘leg-suppressing’’ genes which removed legs from abdominal segments of millipede-like ancestors followed by ‘‘haltere-promoting’’ genes which suppressed the second pair of wings of four-winged ancestors. . . . Each of the wildtype thoracic and abdominal segments has a unique pattern of differentiated structures which constitutes a morphologically defined state or ‘‘level of development’’. . . . The attainment of any level more advanced than [T2] is a stepwise process in which each step requires the presence of a specific BX-C substance. [2507]

According to this model, Ubx enables discs to raise their state from the T2 “baseline” to a T3 level, and abd-A and Abd-B would allow segments to “climb” to higher abdominal states (an idea that is counterintuitive because the latter segments seem simpler). This view of Ubx as a switch chimed with Kauffman’s Binary Code Conjecture [2158, 2159] and Garc´ıa-Bellido’s Selector Gene Hypothesis [1358] (cf. Ch. 4). Garc´ıa-Bellido rightly pointed out that, whatever its nature, the code must be abstract because halteres and legs look nothing alike yet are affected jointly by Ubx LOF mutations. The ability of Ubx GOF mutations to transform wings into halteres (the “Contrabithorax” effect) is consistent with this argument [673, 1547, 2921]. Given the presumption that every T3 cell uses Ubx in the same way (i.e., for its identity [1121]), it came as a surprise when Ubx protein was found to be distributed unevenly in T3 discs (Fig. 8.2) [591]. Ubx’s expression is stronger in the P compartment of both discs, and the most intense subregions are the central (distal) part of the haltere disc and the tibial area of the leg disc [256, 496, 4626, 4627]. Conceivably, this analog heterogeneity might have no functional signiﬁcance if a certain threshold of Ubx is needed for the T3 state. That threshold would be exceeded throughout T3 but not T2. This simplistic rationale is consistent with the fact that T3 subregions are differentially sensitive to Ubx dosage in their tendency to transform [4006]. Another possibility exists. Ubx might be unevenly deployed because it is needed more critically in those T3 areas that differ strongly from T2: (1) the haltere’s bulbous “capitellum” (vs. the huge wing) and (2) the hind part of the 3rd-leg tibia where transverse rows arise (vs. the 2nd leg where there are no such rows; cf. Fig. 3.10). According to this “Use-as-needed Scenario” the anterior cells of the 2nd and 3rd legs would never

244

IMAGINAL DISCS

a Ubx D A

P V

b PS4

PS5

PS6 pedicel

c

Haltere

Wing

capitellum

d Leg 2

A

Leg 3

P

A

T2

P

T3 transverse rows

need to know that they belong to different metameres, so Ubx could safely disappear from those regions over evolutionary time (if it was ever there to begin with). As explained below, this scenario turned out to be correct.

But Ubx does so by directly managing target genes in multiple echelons The ﬁrst detailed model for eukaryotic gene circuitry was proposed in 1969 by Roy Britten and Eric Davidson [473]. They invoked 5 tiers of causally linked DNA or RNA elements called “sensors, integrators, activators, receptors, and producers.” Ever since this “Cascade

Model,” researchers have been predisposed to thinking of genetic control in terms of hierarchies [3773], where genes at one level only inﬂuence genes at the next lower level [98, 659, 974, 2410]. To use a military analogy, the basic idea is that generals would never talk to privates, but rather must give orders to subordinate soldiers through an inviolable chain of command. Ed Lewis made just such an assumption in his Ratchet Model. (Italics are author’s.) The various BX-C substances are presumed to act indirectly by repressing or activating other sets of genes which then directly determine the specific structures and functions that characterize a given segment. [2507]

CHAPTER EIGHT. HOMEOSIS

245

FIGURE 8.2. Expression of Ubx in thoracic disc development. Abbreviations: PS (parasegments), T2 (2nd thoracic segment), T3

(3rd thoracic segment). Axes are indicated by the compass: A-P, anterior-posterior; D-V, dorsal-ventral. See also Appendix 7. a. Fate map of the left half of an embryo (cf. Fig. 4.1), showing the ectodermal region (PS 5–13) where Ubx is expressed (shaded) at Stage 11 (cf. Fig. 4.2). Black ovals mark where discs arise in T2 and T3. b. Expression of Ubx in T2 (wing and 2nd leg) and T3 (haltere and 3rd leg) discs from the left side a mature larva. Degrees of shading signify amounts. Each thick vertical line bisects the discs into A and P compartments (cf. Fig. 4.4). Note that Ubx expression has been altered since the embryonic stage. These changes are likely due to the facts that (1) embryonic and imaginal cis-enhancers are separate at the Ubx locus and (2) the hegemony of the Pc-G or Trx-G complex can be overturned by sufﬁciently powerful trans-acting factors (cf. Fig. 8.1) [3397]. In T2, Ubx persists in the leg disc’s P compartment, but it has vanished from the wing disc except for the peripodial membrane (not shown). A new spot (arrow) arises in the anterior tibial area of the 2nd-leg disc. The 3rd-leg disc has a similar spot, along with an intense P patch that will form transverse rows of the tarsus (d) and tibia. In both T3 discs, Ubx expression is stronger than in the T2 discs, and it is stronger in the P than the A compartment. In the haltere disc, the intensity is also high in part of the A region (arrow) near the center (future capitellum) and in the surrounding fold (future pedicel). c. Derivatives of the haltere disc (pedicel and capitellum). Basal hinge (scabellum) and ﬂank sclerite (hemi-metanotum) are not labeled. d. Bristle pattern of the 3rd-leg basitarsus, showing transverse rows that are unique to T3. Unlike the 1st leg, whose transverse rows are anterior, these rows are on the P side, whereas the 2nd leg lacks transverse rows altogether (cf. Fig. 3.10). Fate map in a is based on [1739]. Data on Ubx expression in the embryo is from [53, 324, 675, 684, 1990], which should be consulted for spatiotemporal subtleties. Ubx expression in the discs (b) is from [496, 4564, 4626]. The latter references are not entirely consistent with one another, and the sum total of the published domains is depicted here. For details of anatomy (c and d) and fate maps, see [17, 526, 1358, 1714, 1883].

In 1998, this paradigm was toppled by Scott Weatherbee et al. in Madison, Wisconsin [4564]. They studied Ubx’s target genes in the haltere disc. Their key ﬁndings are listed immediately below in terms of how Ubx achieves its evolutionarily assigned task to “rewire” the circuitry of T3 development so that a haltere is produced instead of a hindwing. The ability of Ubx to exert opposite effects on scute in different parts of the haltere (row 3 vs. 4 in the table below) implies that it is binding different AS-C cis-enhancers jointly with regional co-activators or

co-repressors [4564]. Direct regulation is also suggested by its cell autonomy, which indicates that Ubx is necessary (LOF) and sufﬁcient (GOF) for control of its target genes. Finally, the regional speciﬁcity of Ubx’s effects within the haltere argues that it is affecting each gene independently of the others. In summary, the “general” in this case (Ubx) is apparently giving orders not only to colonels, but also to lieutenants, sergeants, and privates. This “Micromanager Epiphany” of 1998 [51] has sparked a rethinking not only about Ubx but also about Hox genes collectively:

Problem

Solution

Evidence

Reshape the hindwing into a balloonlike structure (the capitellum).

Ubx blistered (one of the lowest genes in the venation hierarchy; cf. Fig. 6.11). Thus, Ubx prevents the D and V surfaces from annealing. First, Ubx {spalt and spalt-related (salr)} so as to stiﬂe the 2–3 and 4–5 intervein areas [986]. (Ubx does not affect omb – another Dpp target; cf. Fig. 6.3.) Second, Ubx vgQE (cf. Ch. 6). Finally, Ubx wg in the P compartment. Ubx AS-C (a target of Wg). In the P region, Ubx blocks the Wg pathway at its source by preventing activation of wg by Notch: Ubx wg (cf. Fig. 6.8). (Ubx does not affect vgBE – another Notch target.) Ubx AS-C in the pedicel.

In Ubx-null haltere clones, blistered is autonomously derepressed. Conversely, blistered is turned off in UbxGOF wing cells. In Ubx-null clones in the A part of the haltere, salr is autonomously derepressed. Conversely, salr is turned off in Ubx-GOF wing cells. Ubx-null clones derepress vg QE in the “pouch” and wg along the P portion of the D/V boundary. Ubx-null clones derepress scute autonomously in the A part of the haltere’s D/V line, while they derepress wg in the P part. Conversely, scute is turned off in Ubx-GOF wing cells.

Reduce the organ’s size.

Eliminate the bristles along the margin.

Put stretch-sensitive sensilla at the base (pedicel).

Ubx-null clones in the pedicel fail to express scute.

246

1. Dipteran halteres did not arise in a sudden (“hopeful monster”) way [52]. Rather, “the evolution of the haltere progressed through the accumulation of a complex network of Ubx-regulated interactions” [4564]. After Ubx suffused T3 of arthropod ancestors [144, 1618, 3431, 3911], mutations must have created Ubxbinding sites in the cis-regulatory regions of one gene after another [664]. Ultimately, Ubx inﬁltrated enough nodes in the circuitry to reconﬁgure the hindwing into a haltere [3875]. Ubx must have reprogrammed the 3rd leg from a T2 pattern in a similar way [4112], while Scr did the same for the 1st leg [3333], although only a few changes were needed in each case (cf. Scr’s density in the sex comb [1508, 2393, 3285, 4243]; also cf. Fig. 3.10). (This process of “slaves” becoming entrained to new “masters” [1440] is also seen in endocrine systems where hormones evolved before receptors [219, 2969, 4530], and the idea that signals predate the ability of cells to respond was argued by Stern in 1954 [4096]; cf. Ch. 3.) Although T3 evolved gradually, some species could have arisen as “atavistic monsters” via a Ubx LOF defect that reverted T3 to T2 in one step [4635]. 2. Ubx does not “label” the entire metamere [682]. Because Ubx is not acting as a selector gene for every imaginal cell in T3, its on-or-off state need not be clonally propagated like the state of a compartmental selector gene [591, 1547, 2921]. This insight may explain the Collective Amnesia Conundrum (cf. Ch. 4), and it sheds light on why transformed tissue can “revert” to an untransformed state during regeneration [16, 17, 4324, 4326]. Indeed, we can now understand why marginal Ubxnull haltere clones transform nearby Ubx+ cells when the latter are goaded to proliferate [3875] – a heretical effect that violates the dogma of homeotic autonomy [2198, 2421, 2506, 2924, 2925]. Ironically, one compartmental selector gene can overrule Ubx: en GOF clones transform anterior haltere to wing tissue at 100% frequency (n = 65) [1162] due to a paradoxical “En Ubx” link [2670, 2725]. 3. There is nothing sacred about segmental or parasegmental Hox gene expression [682]. Hox genes tend to be expressed parasegmentally in the embryonic ectoderm [2717], but these allegiances can later shift in discs to segmental [2421, 2723, 3930] or idiosyncratic patterns [1168, 2094, 2655, 3285], depending on the need to control target genes in speciﬁc subregions [682, 2717, 2937]. This Use-as-needed Scenario explains why disabling particular Hox genes can have different homeotic consequences at different stages of development [2934, 4671]. For example, haltere cells “read”

IMAGINAL DISCS

their Ubx state at least twice: once to choose a hair morphology for the capitellum and later to select a sensillar type for the pedicel [3623]. Likewise, leg vs. antennal cells read the Antp state during one time window for sensillar identity and during another for the type of limb morphology [2406, 3775, 3810] (cf. additional cases [281, 685]). 4. Cell types are not necessarily encoded digitally [3623]. Conventional wisdom has always asserted that cell types are distinct [3448, 4105, 4508, 4513] because they use sharply deﬁned batteries of genes [473, 3115]. However, varying the dose of Ubx can convert haltere-type cells into wing-type cells through a spectrum of intermediate states [1735, 2921, 3623] (cf. intersexual bristles [1716, 1845, 1883, 2978, 2979]). These intergradations imply that analog logic may be used as often as digital logic in setting the outputs for “realizator” genes [1635, 1807, 2446]. This shift in our thinking about Hox genes actually began in 1995, when James Castelli-Gair and Michael Akam (Cambridge, UK) deftly solved the “3 Genes vs. 9 Segments Paradox” of the BX-C [684, 1121]. That paradox arose in 1985, when Ernesto S´anchez-Herrero et al. showed that the BX-C contains only 3 bona ﬁde genes [3740], despite Ed Lewis’s proof that the BX-C controls 9 different segment identities [2507]. Castelli-Gair and Akam focused on how Ubx speciﬁes parasegment PS5 (= T2p and T3a, where “a” and “p” denote anterior vs. posterior) and PS6 (= T3p and A1a). Their survey of prior models is given below, along with their critiques: 1. “Hox Code Model” [661, 1705, 2156, 2507, 4149, 4695]: metamere identities are speciﬁed by particular combinations of Hox genes. Because only Antp and Ubx are expressed in PS4–PS6, their codes would be as follows, with Antp’s state listed ﬁrst and Ubx’s second in each pair of digits (0 = off, 1 = on): PS4 (10), PS5 (11), PS6 (01). Although consistent with the expression data [661], this model is contradicted by LOF-GOF data (switches are underlined): a. LOF test: Antpnull mutations should convert PS5 (11) to PS6 (01). Instead, they transform PS5 to antenna [4148] or PS3 [4525]. Also, they can cause nonhomeotic defects [1834, 3766, 4148, 4149, 4151], and in subregions of the wing and leg they have no effect whatsoever [4149]. b. GOF test: Ubiquitous expression of Antp should convert PS6 (01) to PS5 (11). Instead, it has little or no effect there [1468, 1550, 4862]. c. GOF test: Ubiquitous expression of Ubx should convert PS4 (10) to PS5 (11). Instead, it transforms

CHAPTER EIGHT. HOMEOSIS

PS4 to PS6 (01) [1549, 2676]. This effect is attributable to a “Ubx Antp” link [1549], which ﬁts two general models for how co-expressed Hox genes interact [648]: (1) the “Posterior Prevalence Model,” wherein posterior Hox genes nullify the effects of more anterior ones in a functional hierarchy [1007, 1113, 1549, 1550, 2646], and (2) the “Hox Competition Model,” wherein Hox proteins compete for DNA binding sites [89, 685, 2396, 4806]. While thoracic discs may not use a combinatorial Hox code, the labial and eye discs do appear to employ a heteromeric Hox protein complex for this purpose [3333]. 2. “Hox Isoforms Conjecture” [256, 2371, 4197]: Ubx exons are spliced differently to dictate PS5 (isoform type 1) vs. PS6 (isoform type 2). The following facts refute this notion: a. Expression of the different Ubx isoforms is not metamere speciﬁc [115, 2308, 2600, 3156]. b. LOF test: Mutations that eliminate speciﬁc Ubx isoforms affect metameres uniformly [583]. c. GOF test: Ubiquitous expression of particular Ubx isoforms affects metameres uniformly [684, 2676, 4197]. 3. “Hox Threshold Conjecture” [1549, 2421]: different amounts of the same Hox protein specify different metameres. The authors discuss the prior evidence for this idea and conclude that alternative explanations exist in each case. Their own data are most easily explained by assuming that (1) anlagen within each metamere respond to Ubx independently of one another and (2) they do so by using a single (on/off) threshold. That is, the logic is digital. In wild-type embryos, certain anlagen in PS5 develop like those in PS4 (where Ubx is off), while others mimic PS6 (where Ubx is on). Ubx must be instrumental since disabling it (Ubx null embryos) forces both PS5 and PS6 to look like PS4 [1772, 4152]. What Castelli-Gair and Akam found is that the PS6-like rudiments of PS5 express Ubx during the periods when their destiny is being decided. Evidently, Ubx is acting like a paintbrush to give them a PS6-like state. Ubx’s dynamics are thus “not simply transitional stages to reach a mature pattern of relevant gene expression.” Rather, “they are fundamental for the normal function of the gene.” The importance of temporal regulation is obvious for the 3rd-leg disc, which is permitted to arise by the absence of Ubx at an early stage (Ubx-0ff allows Dll-on) but then is modiﬁed by uneven deployment of Ubx at a later stage (Fig. 8.2). The authors proposed a model that is tantamount to the Use-as-needed Scenario:

247

According to our model, the unique character of a segment depends on its temporal and spatial pattern of Hox gene expression. This is its ‘‘Hox code’’. For a single cell, the Hox proteins are just like any other transcription factors. What they will do depends on the context in which they are expressed, and need not always have the same consequences in terms of ‘‘segment identity’’. . . . In limb or wing primordia, it may be more useful to think of Hox proteins as defining the difference between specific alternative developmental pathways (wing trichome vs. haltere trichome) than to think of them as specifying global segment identity. [684]

Elsewhere, Castelli-Gair has rightly pointed out how (1) this cellular (vs. segmental) role for Hox genes makes sense evolutionarily, and (2) we have been deluding ourselves with our own terminology. Hox genes are not controlling segment identity, but cellular behavior that will result in a certain segment morphology. This is what Hox genes do in unsegmented organisms like C. elegans [3727] and is probably what they did in the common ancestor of all metazoans. Segment identity is a subjective concept that originates from the observation that in a particular species, a number of cell characteristics are always associated in a given segment. [682]

In conclusion, it seems that the Hox genes not only work like micromanagers but also have been assigned a fairly heavy “work load” during evolution [968, 1114, 4563]. They govern particular downstream genes [1591, 3457] on a cell-by-cell basis [682], although cells of like kind may be transiently organized metamerically during an early stage of development [4893]. As Akam poetically put it: Some transcription factors appear to be specialists: they specify a particular fate or behavior whenever they are expressed in a cell; the myogenic factors might approximate this role, for example. The Hox gene products lie at the other extreme: they are versatile generalists. They operate in many different cell and tissue types, where they modulate, sometimes dramatically but more often subtly, a wide range of developmental processes. In each of these cell types, expression of a Hox gene means something different -- to divide or not to divide, to make or not to make a bristle, to die or not to die. In any given lineage, that meaning probably changes several times during development, in response to hormonal and other developmental cues. [51]

Pc-G and Trx-G ‘‘memory’’ proteins keep homeotic genes ON or OFF. Although the expression patterns of Hox genes are modulated in certain discs, the gross layout of their metameric domains is retained throughout development. How is this done? That is, when the segmentation gene hierarchy turns on a speciﬁc Hox gene in a particular metamere (cf. Fig. 4.2), how does that gene manage to stay on after the segmentation machinery fades away?

248

This question echoes the old “Memory Riddle” in embryology [2417, 2501] – viz., how do cells maintain particular states of determination from the stage when they acquire them until the stage when they express them [1365, 2755, 3882]? That riddle has been especially troubling for imaginal discs, where the bracketing stages (embryogenesis and metamorphosis) are separated by several days but can be extended (by serial transplantation) to months or even years [1668, 2755]. One solution proposed by Wolpert is that positional “values” of some kind (digital?) perpetuate the transient (analog) positional information (cf. Fig. 4.3) [4724]. Theoretically, the simplest way for a gene that encodes a transcription factor to remain on is for it to activate itself in a feedback loop [1790, 2324]. Some Hox genes employ this strategy [1429, 2372], whereas others use an indirect route [321, 325, 1952, 4312]. However, autoregulation is the exception rather than the rule in the ectoderm [682]. In the imaginal discs, the on or off states of most Hox genes are sustained by chromatin-remodeling proteins. Most of those proteins fall into two groups [3197, 3275, 3394] – one named after Polycomb and the other after trithorax. (See [482, 849, 1490, 2598, 3868] for shared factors.) These groups regulate not only the Hox family but also compartment selector genes, including apterous (D/V) [3752] and engrailed (A/P) [3512] . LOF mutations in the Polycomb Group lead to the turning on of homeotic genes wherever they are normally off, while LOF mutations in the Trithorax Group lead to the turning off of homeotic genes wherever they are normally on. (Ubx may also regulate itself via a negative feedback loop [1989].) Proteins in the Polycomb Group (Pc-G; ≥14 members [3395] ) form a variety of multiprotein complexes [3453, 4315] . The complexes self-assemble via several types of interaction domains [2373, 2374, 4313] . Pc-G complexes bind speciﬁc “PRE” (Pc-G Response Element) motifs [1902] at ≥80 polytene chromosome sites [3078] , including the BX-C and ANT-C [2622] , where they stabilize the nucleosomal structure of inactive chromatin [455, 3869] . For this purpose, some Pc-G ensembles use histone deacetylation [4315] , but others may not [3869]. Most important, Pc-G proteins stabilize the off states of BXC [2772] and ANT-C genes [1491, 4026] during early embryogenesis [3452] and preserve those states throughout larval development [584] . In other words, they ensure “the clonal transmission of a determined state” [1026], although the states are only “ﬁrm biases” [3794] that can be overruled later. Different subsets of Pc-G proteins govern different tissues [3453] , due, in part, to regional feedback from target genes (e.g., “Ubx Pc”) [3276].

IMAGINAL DISCS

Pc-G genes can mimic position-effect variegation [3452] , suggesting that they use the same silencing pathway as heterochromatin [388] . Indeed, there are some

shared components [2622, 2966], but in general the route is different [549, 1199, 3968]. Most Pc-G genes (e.g., Polycomb) are named for sex combs (cf. Table 8.1) [2189] because LOF mutants have extra combs. This phenotype stems from the fact that (1) Pc-GLOF mutations lead to expression of Sex combs reduced (Scr) in subregions of T2 and T3 (in violation of the Posterior Prevalence Rule [1113]), and (2) Scr then forces a sex comb identity on the basitarsi. Such mutants thus have the normal number of legs, but all six legs look like forelegs – that is, like a man with arms where his legs should be. Proteins in the Trithorax Group (Trx-G; ≥ 15 members [1490] ) also form a variety of chromatin-remodeling complexes [2114] . Like Pc-G complexes, the member proteins associate via various interaction domains [3666] . Trx-G complexes bind many of the same polytene chromosome bands as Pc-G complexes [1436] , and “TREs” (Trx-G Response Elements) have been mapped to the same restriction fragments as PREs [3196] . However, in the one case analyzed in detail (Ubx upstream region at ∼10 b.p. resolution), the TREs and PREs do not overlap [4319], nor has any direct interaction between Pc-G and Trx-G complexes yet been found [3260]. Trx-G complexes sustain the on states of genes in the BX-C [719] and ANT-C [4468] after embryogenesis [451] in a tissue-speciﬁc [454] and time-sensitive [3847] manner. In contrast to Pc-G complexes, Trx-G complexes tend to stimulate transcription [848, 2114] by remodeling chromatin so as to make DNA more accessible to transcription factors [4892]. To do so, some Trx-G ensembles acetylate histones [203] – the same trick that is used by dosage-compensation complexes to double the transcriptional output of X-chromosome genes in males [1633] . Other ensembles may displace nucleosomes [4377] via ATP-driven, mechanochemical reactions [1700] . Our current view of how Pc-G and Trx-G complexes work as memory devices [2981] can be illustrated by again considering the expression of Ubx in PS4 (off) vs. PS6 (on). The differential regulation in this case is thought to involve least three distinct phases (Fig. 8.1b) [719, 3196, 3395, 3397]: 1. The segmentation gene hierarchy (SGH) turns Ubx on in PS6 (by deploying an excess of trans-activators there) and off in PS4 (by deploying an excess of transrepressors there). The impacts of these transcription

CHAPTER EIGHT. HOMEOSIS

factors are local, transient, and interactive [323, 1712] (e.g., via quenching [1600, 1601]). 2. SGH activators recruit Trx-G complexes to a nearby TRE site, while SGH repressors recruit Pc-G complexes to a PRE site [2171, 3395, 3396], thus “bookmarking” the DNA as to which regions were being read or not read at that time. In both cases, the assembly process appears to be stepwise, with different subsets of proteins being deployed in turn to execute a series of chromatin-remodeling alterations [3101, 4315]. For example, Extra sex combs (Esc) is transiently needed when the Pc-G assumes command of the Ubx promoter at ∼4 h AEL [3939, 4160], and Esc’s 7-bladed propeller of WD loops may provide the hub for assembling the nascent Pc-G complex [1734, 3102, 3394, 4313, 4314, 4316]. Moreover, separate subsets of Pc-G proteins appear to implement (1) the bookmarks and (2) the transcriptional silencing machinery that is recruited to the bookmark [306]. 3. SGH activators and repressors disappear as Pc-G and Trx-G assume command. In PS4, the Pc-G complexes catalyze a spreading of the “closed” state [4889] over ≥10 kb [3275, 4172], thereby silencing the nearby imaginal disc enhancers [3397, 3938], and those enhancers remain dormant unless awakened later by regional trans-activators [37, 690, 2227]. Spreading might occur via (1) iterative recruitment of histones or (2) looping of the DNA caused by adherence of Pc-G aggregates to one another [4448] or to the nuclear matrix [1393, 4690] . In PS6, the Trx-G complexes tend to keep the nearby imaginal enhancers “open” to either positive [501] or negative [3100] trans-regulatory inputs [675, 3396, 4199, 4378, 4413]. Those later inputs would explain why Ubx’s pattern in discs differs from the embryo (Fig. 8.2). We still do not know how the open or closed states of chromatin are perpetuated so durably during repeated cycles of DNA replication [306, 1317] . DNA methylation cannot be the answer here. It is used widely in mammals [1326] but less so by 1 to 2 orders of magnitude in ﬂies [1589, 2625, 4411] (cf. its link to histone deacetylation [2521]). Also unclear is whether transdetermination is due to a “memory loss” of Pc-G or Trx-G proteins from homeotic gene promoters over prolonged periods of proliferation [849, 2082] (cf. Fig. 6.9d).

Homothorax, Distal-less, and Spineless specify leg vs. antennal fates We may call one thing a wing and another a leg, but the key question is whether the cells themselves “know”

249

that they belong to one vs. the other type of organ [2411, What does “wingness” or “legness” mean to them? Homeotic mutations have allowed us to interrogate ﬂy cells in this regard, but their cryptic answers have taken awhile to decipher. Wolpert argued that when two organs use the same morphogens (e.g., Hh, Dpp, or Wg), they must differ in their mode of interpretation [4724], and Stuart Kauffman proposed that those modes could be encoded by on/off states of Hox genes [2155, 2156]. For three decades, this view prevailed [2755]. In 1998, the same Micromanager Epiphany that toppled the Cascade Model also challenged this Hox Code Model [51]. To wit, why should Ubx be unevenly expressed and variously employed in haltere cells when it is only supposed to label cells with T3 identity [51, 684, 3875, 4564]? The antenna-leg dichotomy brings this issue into sharper focus. Like the haltere and wing, the antenna and leg are serially homologous [3447], and they use the Hh-Dpp-Wg signaling circuitry in similar ways [1037, 1833, 2082, 4277]. However, the antenna does not normally express any Hox gene [1168, 2103, 2167, 3527], so it is unclear how it manages to develop differently from a leg, which is considered to be the ground state [613, 677, 2936]). After the four-winged Ubx LOF ﬂy, the next most famous homeotic mutant has legs where its antennae should be, hence the name “Antennapedia” [1468]. In Antp GOF ﬂies, Antp is ectopically on in antennae [2094, 4806] aside from its normal, albeit uneven, expression in leg discs [677] . Based on this result alone, it would seem that the ectopic Antp is “instructing” the antennal tissue to adopt a leg fate [1416]. However, virtually any Hox gene can turn antennae into legs when forced on there (Table 8.1), so Antp’s effect is actually nonspeciﬁc. This effect is due in part to disabling of the Eyeless protein [3402]. It also involves an inhibition of the genes homothorax (hth) [677, 4806] and spineless (ss) [1119], both of which encode non-Hox transcription factors. Hth is a homeoprotein [2360, 3226, 3589], and Ss is a bHLH-PAS protein [1119, 1166]. 3794].

{Antp or other Hox gene}

{hth and ss}.

The etiology would be simple if hth and ss were expressed only in antennae (i.e., they could dictate antennal identity directly), but they are both expressed in leg discs also (Fig. 8.3; cf. Figs. 5.11 and 5.12) [9, 130, 677, 1119, 2673]. To state the dilemma more succinctly, how can genes a and b, expressed in both discs C and D, make disc C different from disc D? In theory, this “Shared Genes

250

IMAGINAL DISCS

antenna

a Homology map

A1

Ar

A2 A3

ro

distal

xi m

al

Cl

p

Co

BC T5

T4 T3 T2

Tr

T1 Ti

Fe

b Either Duo Hypothesis 7 6

Gra

[M]? 5

xi m

ro

distal

al

1. Specification

p

2. Interpretation AND early

Hox

t?

7 6 5 4 3 2 1 A3

Ar BC early

hth Dll spalt ss x? dac bab al

dien

4 3 2 1

A-type? A2

A1

leg

positional values?

L-type? ClT5T2-4 T1

Ti

Fe Tr

Co

? segment code?

Hth and Dll early

Ss X?

or

A-type

and

A' B' C' D' E' F' G detailing?

Co?

segmental

A B C D E F G identities? detailing? pattern

Ar BC

A3

A2 A1

antenna

x T5 T2 T1 Ti Fe Tr Co Cl T3 T4

leg

CHAPTER EIGHT. HOMEOSIS

251

FIGURE 8.3. Our current understanding of what makes an antenna different from a leg. This problem was discussed earlier with

regard to the 1969 debate between positional information (PI) and prepatterns (cf. Fig. 4.3). It is revisited here to see how much progress has been made since then. The answer, in short, is “a little.” a. The old problem posed by the 1:1 correspondence of antennal and leg regions. These homologies were deduced from Antp GOF phenotypes [3445] and conﬁrmed in other mutants (e.g., bab GOF [1516]). b. The “Either Duo Hypothesis” for why antennae develop differently from legs. Antennae and legs are both thought to use an unknown morphogen (M) to specify radial positions relative to the future tip (cf. Fig. 5.4). In keeping with Wolpert’s model, cells may convert this scalar signal into digital states (“positional values”), but those states are not propagated by cell lineage [544, 3446, 4575], nor are they stabilized by “memory” genes of the Pc-G or Trx-G (see text). Thus, no “recording” phase is interposed between speciﬁcation and interpretation. (A and L denote antennal vs. leg types of development.) Eventually, the antennal and leg rudiments turn on the same region-speciﬁc genes but express some of them differently. Horizontal bars denote expression domains; gray shading means weak or incomplete expression; checkered shading indicates heterogeneity. (N.B.: the axis is reversed relative to Fig. 5.11.) One code is well documented: the combination “hth-on and Dll-on” (1) turns on spalt and (2) evokes ectopic antennae in other discs (via Gal4 drivers) [1085]. Also, hth LOF or Dll LOF clones turn off spalt and cause antennal-to-leg homeosis [677, 1085, 3226, 3380,3716]. The implication is that antennal identity is encoded by the Hth-Dll overlap, which leg discs lack (until late-3rd instar) [4760]. If so, then how do cells outside A2–A3 avoid making leg tissue? Distally, this role may be ﬁlled by ss because ss LOF clones exhibit the same homeosis as hth LOF or Dll LOF [564, 1119, 1166, 2933, 4150, 4508]. However, ss GOF cannot induce antennae outside the head (except for a minor claw-to-arista switch in the leg) [1119], so an unidentiﬁed factor “X” (in antenna but not leg) probably cooperates with Ss as Hth works with Dll. Either of these pairs of agents could enable antennal cells to deviate from the leg program in the same way that Ubx lets haltere cells deviate from the wing program (cf. Fig. 8.2). The logic is summarized in the circuit diagram (cf. Fig. 2.7 for symbols), which also shows how exogenous Hox proteins (e.g., Antp) cause homeosis by stiﬂing Hth [4806] or Ss [1119]. The ﬁnal stage of development involves creating patterns of small cuticular elements (e.g., bristles and sensilla, depicted abstractly here). We know a fair amount about how this is done [1653, 2055, 3531, 3548, 4125] (see Chs. 3 and 5 for the leg), but the links between the genes that act locally and the ones that act at a larger scale remain elusive. Those links may involve an intermediate level of prepatterns (cf. Fig. 6.14), so Stern’s hypothesis is still as relevant as Wolpert’s (cf. Fig. 4.3). Overall, this system is murkier than our picture of embryo segmentation (cf. Fig. 4.2). Remaining questions include: (1) What is the initial bias that steers tissue into an A- vs. L-type mode of interpretation? and (2) Is A1 actually a “coxa in disguise,” or does it use a third duo of genes to veer away from a leg fate? Black or white symbols in the antennal and leg schematics are abstract renditions of the actual patterns. Genes (“DO” = details omitted): al (aristaless) [618, 881] , bab (bric a` brac) [1516], dac (dachshund) [4761], Dll (Distal-less; DO: expression above the tibia only appears in late-3rd instar) [1085, 3242, 4761] , hth (homothorax) [1085, 4761] , spalt [1085] , ss (spineless; DO: expression in leg disc shifts to periphery in late-3rd instar; cf. tango) [1166] . In the leg disc, spalt is expressed in “isolated cells scattered around the proximal region” [220]. N.B.: During early antennal development, hth is expressed throughout the primordium [677], and ss expression is dependent on hth and Dll (I. Duncan, pers. comm.). This link is drawn as a dashed line in the circuit diagram (near the bottom of the ﬁgure). The diagram in a is redrawn from [3445], and domains of gene expression (b) are modiﬁed from [3242] based on the above references.

Riddle” can be solved simply by combinatorial logic (cf. the Venn Overlap Rule, Fig. 6.10): 1. Genes a and b could jointly specify identity “C”. 2. They would both be expressed throughout disc C. 3. However, they would be expressed in nonoverlapping subregions in disc D, thus leaving all of D’s cells in a default state (e.g., leg vs. antenna). For hth, the riddle was solved in 2000 by Si Dong et al. in Madison, Wisconsin [1085]. There are two other homeobox (but non-Hox) genes that are important in the story: extradenticle (exd) [3319, 3526--3528, 3715] and Distal-less (Dll ) [836, 4212]. Indeed, the original name for Distal-less was “Brista” – a contraction of “Bristle on arista” (referring to the antenna-to-leg transformation) [2561, 4212].

1. Hth-Exd dimers are needed in the nucleus for antennae to develop differently from legs [677]. These dimers normally occupy the entire hth-on domain because exd is on in all hth-on cells (indeed in all antennal and leg cells) [8, 9, 130, 677, 2673, 3589]. Thus, hth LOF or exd LOF clones cause an antenna-to-leg transformation on a cell-by-cell basis. 2. Ectopic expression of Hox genes inhibits transcription of hth (e.g., Antp hth) [677, 4806] and hence prevents Extradenticle (Exd) from entering the nucleus [156, 3332, 3527, 3589]. 3. In the antennal rudiment, the overlap between hthon (proximal) and Dll-on (distal) domains is substantial, but in the leg disc it is nonexistent until late-3rd instar, and even then it is minimal (Fig. 8.3). The condition “hth-on and Dll-on” must dictate

252

IMAGINAL DISCS

antennal identity because (1) Dll LOF alleles partly transform antennae to legs [838, 1085, 4212] and so does hth LOF [677, 3226, 3380, 3716], (2) ectopically expressed Dll induces antennae in places where hth is normally on and vice versa [1085, 1561], and (3) co-expression of Dll and hth via Gal4 drivers induces ectopic antennae in legs, eyes, head capsule, and genitalia [1085]. The susceptibility of the genitalia makes sense because, like antennae, they evolved from legs [1179, 1562]. Interestingly,“hth-on and Dll-on” turns on a speciﬁc target gene – namely, spalt [1085], although spalt alone does not dictate antennal identity (cf. Ch. 6). The Boolean logic of the hth-exd-Dll synergy could be implemented by a Hth-Exd-Dll complex that only activates antenna-speciﬁc genes when all three jigsaw pieces ﬁt together [1085, 3242]. Indeed, the proteins do associate in vitro [784]. This complex must function like Ubx alone in the haltere (i.e., it allows antennal cells to depart from a leg fate in various ways). For ss, the situation is less clear. LOF alleles transform the distal portion of the antenna into tarsal structures [426, 564, 1119, 1166, 2933, 4150, 4508], but ectopic GOF expression evokes only a paltry conversion of claws to aristae [1119]. This disparity may be due to an essential cofactor “X,” whose gene is expressed throughout the ss-on domain in the antenna but not the leg (Fig. 8.3). Tango is a dimerization partner for Ss [1166], but it does not appear to solve this puzzle because it is so broadly expressed in both antennae and legs. As a working hypothesis, the overall rule for antennal “identity” can be phrased as an “Either Duo Hypothesis”: {Hth-Exd and Dll} or {Ss and X} cell behaviors.

antenna-speciﬁc

This control circuit may be in two parts because it evolved that way [1119]. Moreover, evolution may have spared the basal “A1” segment, which expresses neither Dll nor Ss (Fig. 8.3). That annulus may thus retain a leg style of development, just as parts of the 3rd leg seem to have never been steered away from a 2nd-leg fate by Ubx (see above). Whatever the nature of these proximaldistal zones, they are certainly not clonal [544, 3446, 4575], so any biases they impose must be transitory and changeable via mitosis [3448, 3794] (cf. Cabaret Metaphor, Ch. 4). The sensitive period when antennal cells can be diverted to a leg fate is early-3rd instar [1468, 1623, 3286, 3333, 3445, 3775], just after spineless is turned on by Dll [1119]. Remarkably, the homeotic leg can come from as few as 10 transformed antennal cells [3445].

If a ‘‘master gene’’ exists for the eye, then it is also a micromanager It is hard to imagine a stranger creature than one with eyes on its legs and wings, but ﬂies with just this anatomy were reported in 1995 by Georg Halder et al. in Basel [1685]. Halder et al. created these monsters by turning on the “Pax” (Paired box) gene eyeless (ey) in discs where it is normally not expressed. Although ey has a homeobox in addition to its paired box [3486], the ey LOF phenotype is unlike any Hox LOF ﬂy insofar as it involves an absence, rather than homeosis, of the affected structure (hence the name “eyeless”). The ability of ey GOF to turn on all the downstream machinery needed to build an eye implied that ey must be the “master control gene” for eye development [214, 1415, 1416]. Ironically, Stern and Tokunaga had hailed an allele of this same gene (ey D ) as their long-sought “prepattern mutant” ∼30 years earlier (cf. Ch. 4) [4109]. Further research, however, revealed several caveats that dampened the enthusiasm for the master gene concept [3658]: 1. The homeotic effect is dependent on the tissue context: ey can only induce ectopic eyes in certain dppon zones in speciﬁc discs [394, 744, 1684, 1685, 3894]. Ectopic expression of dpp alone does not evoke homeosis in any disc [2755]. 2. Other genes besides ey were found to also have the power to elicit ectopic eyes [1234, 1419, 4385], including eye gone (paired/homeobox) [1780, 2090] and eyes absent (eya) [394, 398]. Surprisingly, two such genes are normally on in other discs as well: dachshund (dac) [3894] and teashirt (zinc ﬁnger) [3240] (cf. sine oculis in the leg disc [3118]). In theory, these other genes need not undermine ey’s primacy because they could be acting downstream. However, several of them can act upstream (in a feedback loop) to turn ey on at ectopic sites [743, 3387, 3894]. 3. Still another homeobox gene optix can elicit extra eyes, and it does so completely independently of ey (i.e., in an ey null background) [3851]. The growing disenchantment was summarized by Pan and Rubin in 1998 [3240]. (The homeobox gene sine oculis is abbreviated “so” [759, 3859].) Although ey is required for the initial expression of eya, so, and dac in the eye primordium, the later genes are also involved in a positive feedback loop to activate the expression of ey. Therefore, ey does not function simply as a ‘‘master regulatory gene’’ to activate a linear pathway specifying the eye fate; rather, ey, eya, so, and dac form part of a regulatory

CHAPTER EIGHT. HOMEOSIS

253

network that together ‘‘locks in’’ the eye specification program. [3240]

Then, in 1999, an ey paralog called “toy” (twin of eyeless) was described [933], which acts upstream of ey (Toy ey) to induce ectopic eyes [1419, 1760]. Unlike ey, toy is not subject to any feedback regulation by dac, eya, or so. Hence, the title of “master gene” was transferred from ey to toy. The anointing of toy may have been premature, however, because we still do not know (1) its null phenotype, (2) its relationship to optix, (3) whether it can induce eyes with drivers other than dpp-Gal4 (which may have its own idiosyncrasies) [1030, 4385], or (4) why it is able to induce ectopic eyes where ey cannot [3118]. If there were a hierarchical chain of command with toy or ey at the top, then co-expression of ey with one of its subordinates would not be expected to manifest much synergy because ey would have to act through them, but strong synergistic interactions (with regard to inducing extra eyes) have been documented for a number of combinations [930, 1030], including {ey and dac} [744], {ey, dac, and dpp} [744], {ey and dpp} [744], {ey and eya} [394, 744], {eya and dac} [743], and {eya and so} [553, 744, 3387]. The relationships among these genes at the inception of eye development are complicated [1787]. As discussed in Chapter 7, several are regulated by dpp, but ey is not [744, 930]. The links below have been demonstrated under endogenous or ectopic conditions, but there is no guarantee that the circuitry behaves similarly in the two contexts [4385]. Evidently, these genes operate as an interactive network rather than as a hierarchy (cf. Fig. 7.3) [3240, 3387]. Link 1:

Link 2:

Link 3:

{dac, eya, and so} during normal Dpp furrow initiation. In Mad null clones at the P edge of the eye (where ommatidial patterning begins), the cells stop expressing dac, eya, and so [930], but this link ceases to operate once the furrow gets underway [930]. Dpp is not needed for ey to turn on [744]. Ey {eya and so} during normal furrow initiation. Neither eya nor so is expressed in ey LOF discs [1684]. Conversely, ey turns on normally in eya LOF or so LOF discs [1684, 3387]. {Ey and Dpp} {dac, eya, and so} when targeted to the wing disc [744]. Expression of ey in dpp-on regions of leg and wing discs (via dpp-Gal4:UAS-ey) activates dac [3894], eya [394, 1684], and so [1684, 3118]. For the eya [554, 4888] and so [3118, 3476] loci, this activation has

Link 4:

Link 5:

Link 6: Link 7:

Link 8:

been shown to occur via eye-speciﬁc cisenhancers. The need for Dpp is shown by ey’s inability to activate dac, eya, or so in dpp LOF eye discs when overexpressed there (via ey-Gal4:UAS-ey) [930]. Induction of eyes by ey also requires Hh [2126]. Eya {dac and so} during normal furrow initiation. When Dpp signaling is disabled (in dpp LOF discs or Mad null clones), expression of dac and so vanishes but can be rescued by overexpressing eya (via dppGal4:UAS-eya) [930]. This talent is not shared by dac or so: neither gene (when expressed via dpp-Gal4) turns on any other members of the eya-dac-so trio [930], nor is so alone able to induce extra eyes [930, 3387]. Likewise, eya LOF eye discs fail to express dac above a trivial level, while dac LOF eye discs still express eya at normal levels [743]. Indeed, the only hint of a “Dac eya” link is a spot of eya in dpp-Gal4:UAS-dac antennal anlagen [743] that is more diffuse than the dac spot induced by dpp-Gal4:UAS-eya. (Hence, no “dac eya” link is shown in Fig. 7.3.) Eya dpp during normal furrow initiation. Expression of dpp is reduced in eya LOF discs and is eliminated entirely in clones that are homozygous for a strong LOF allele (eya1 [553]) [1780]. {Eya and Dpp} dac when targeted to the wing disc [744]. {Eya and Dpp and So} dac when targeted to the wing disc [744]. Within the eye disc, eya LOF and so LOF clones fail to express signiﬁcant amounts of dac [3387]. {Eya or Dac or [So and Eya]} ey in the antennal region of the eye disc [743, 3387, 3894]. However, this area appears to be the most sensitive to eye induction, so these effects are relatively weak and may not be physiologically signiﬁcant.

Like Ubx, Toy and Ey appear to act as micromanagers for genes that are far downstream (as do Eya and So [3387]). Indeed, this fact alone refutes the notion of a hierarchy [4385]. The best example is the regulation of rhodopsin genes in photoreceptor cell types (cf. Ch. 7) [1272, 3895, 4600]. Within ∼70 b.p. of the transcription start site, all rhodopsin gene promoters have a generic binding site for Ey or Toy (or both), which allows the genes

254

to be activated throughout the eye [3256] (cf. the Glassbinding site [1157, 2963, 2964]). More distally, they each have cis-enhancers that are speciﬁc for each rhodopsin subtype (e.g., Prospero represses these enhancers at rh5 and rh6 in R7 [860]). A promoter can only turn on its gene if both the proximal AND distal sites are occupied. Thus, the promoter is like a door with two locks: only when both keys are turned will it open [1705]. Indeed, this sort of micromanagement makes even more sense for a ﬁeld-speciﬁc gene like ey than it does for a metamere-speciﬁc gene like Ubx. Given that Hh, Dpp, Wg, Delta, and Spitz are used by virtually all discs (and in different parts of the same disc in some cases), the cells of each ﬁeld need to have some way of knowing that a certain morphogen signal “means” a certain fate that is appropriate for their ﬁeld [4385]. The meaning would be encoded grammatically by a combination of inputs from (1) a ﬁeld-speciﬁc selector gene and (2) a particular signaling pathway [1477, 1686]. For example: you are in the eye your Ras is tickled at a certain time, THEN become such-and-such a cell type. IF

AND

The former condition could be satisﬁed by ey. In general, ﬁeld-speciﬁc genes appear to function as “licensing agents” for parochial pathways [871, 4563, 4857]. Ergo, the meaning of “wingness” or “legness” would be a certain transcription factor (e.g., Toy in eyes) or set of such factors (e.g., {Hth-Exd and Dll} or {Ss and X} in antennae) that identiﬁes each cell as belonging to that particular ﬁeld. Cells would know their histotypic region of residence, although they might not know their disc per se. This surmise helps explain (1) why homeosis and transdetermination affect histotypes as well as metameres (Table 8.1; cf. Fig. 6.9d) and (2) why development can be perturbed to produce phenotypes that look grotesque to us (e.g., triplicated limbs; cf. Fig. 5.2) but feel natural to the cells themselves because no local rules are broken [617] (e.g., Bateson’s Rule).

The manifold ‘‘enhanceosome’’ is a wondrous Gordian Knot The synergy among early eye genes described above is partly attributable to a ≥3-protein complex wherein Eya binds Dac and So [553, 743, 3387]. Within the complex, So apparently functions to bind DNA [759, 3859], while Dac supplies a transcriptional activation domain [743], and Eya forms a bridge between them [4385], although a different architecture is used in other tissues [2367], and So recruits

IMAGINAL DISCS

other partners as well (including Groucho, Amos, and TAF250 [2193]). The sad fact is that we cannot now solve either the Eya-Dac-So puzzle or the Hth-Exd-Dll puzzle discussed above, nor the riddle of how these aggregates dovetail with the transcription machinery so as to regulate their target genes. In general, humankind is still at a kindergarten level of understanding with regard to “promoter logic” [393, 968, 2836, 4165, 4755]. We would like to learn the following: 1. How do the various transcription factors and cofactors ﬁt together to form “enhanceosomes” [649, 2109, 4274, 4304, 4454]? One example of a recent insight (in ﬂies) is that Hox proteins use their hexapeptide motif to dock with the TALE domain of Extradenticle [879, 2207, 3281, 3393]. 2. How does each enhanceosome dovetail with the basal transcription apparatus [1127, 1317, 2304, 2488, 2826], and to what extent does the gigantic Mediator complex assist this process [1703, 2665]? Virtually all ∼20 subunits of the human Mediator complex are conserved in ﬂies [412, 4386], and the ﬂy complex binds a bewildering zoo of transcription factors – including ¨ Bicoid, dHSF, Dorsal, Ftz, Kruppel, and VP16 (but not Twist or Hunchback) [3262]. 3. How do the steric rules implement “AND” or “OR” conjunctions [1477, 4042], “NOT” negations [1984, 2350], and “IF/THEN” contingencies [440, 464, 845, 2678, 4164]? 4. How are these logical conditions concatenated grammatically [693, 876, 960, 1046, 1444] so as to dictate criteria such as “IF factors A AND (C OR D) but NOT B are bound, THEN transcribe the gene at rate r” [103, 259, 2471, 3657, 4836, 4837]. It is at this level that proteins literally compute outputs, and it is here that we will undoubtedly ﬁnd the key to how genes compute anatomy. Arguably, this “Enhanceosome Puzzle” [1317, 1696, 2304, poses the greatest challenge for the postgenomic era of biology [968, 4847]. Will computational genomics alone succeed in untying this tangled Gordian Knot? No one knows. What we do know is that classical genetics has been incisive in cutting to the core of similar problems. Explorers of the future might therefore do well to study the past since those old tools may yet prove useful [410, 463, 1364, 2845].

2488, 4332]

The deepest enigma is how evolution rewired the circuit elements It has become trite to say that development is like a computer program [2179]. Clich´e or not, there is no better

CHAPTER EIGHT. HOMEOSIS

metaphor for gene circuitry [968, 4837]. In their 1997 book Cells, Embryos, and Evolution, John Gerhart and Marc Kirschner argued that evolution works like an electrician [1440]. By changing the wiring here or there, it reconﬁgures circuits to alter anatomies. Collectively, the many “ ” and “ ” links in the present book have sketched an elaborately ornate wiring diagram for disc development. The deeper question is: where did it all come from? Over the eons of evolutionary time, the repertoire of genes and cis-enhancers must have been relatively stable because of their interdependence [968] (e.g., see [2610, 3601, 3895, 4777]; cf. the genetic code [1148]). Hence, they make up the genomic “hardware” [103]. In contrast, the links among these circuit elements were more changeable [3781] (e.g., see [1617]), so they make up the “software” [694, 2236, 3494]. For example, evolution has rearranged macrochaetes by adding or deleting cis-enhancers at the AS-C (cf. Fig. 3.4) [4766], and dipterans reﬂect this tinkering [662, 1354, 2959, 3966, 4096]. What has changed is not the genome’s overall inventory of available genes or cisenhancers, but rather the choice of which enhancers reside in the AS-C. Linkage in metazoans represents a kind of easily modifiable software of development, which owes its success to the highly conserved hardware of basic cell biological mechanisms. [1440] (p.142) Evolution is about teaching old modules new tricks. [1094]

255

Similar games were undoubtedly played within enhanceosomes themselves. For example, gene “a” encoding a trans-activator for gene “b” (a b) could mutate so that its protein acquires a WRPW motif that now recruits Groucho and turns it into a repressor (a b). Pangolin may be a case in point because it can bind either Armadillo (Pan target gene) or Groucho (Pan target gene). The implication is that evolution kept Pan in a schizophrenic state to serve as a toggle switch (cf. Fig. 5.6c). Other bipolar regulators may include Dorsal [1110, 1257], Mad [3377], NK-4 [776], Su(H) [1330, 2540], and Tramtrack [2391]. The making and breaking of connections is what nervous systems do as they learn, so neural networks may offer a useful way to simulate this process for gene circuits in the future [133, 297, 441, 1438]. One tantalizing question is whether living systems have “learned” to increase their “evolvability” [2154, 4520] by making certain types of genomic alterations easier than others [622, 1090, 3115, 4798] (e.g., transposition [420, 2212, 4632]). For developmental biologists, the pithiest issue remains how the genes encode the anatomy. Do ﬂies really have a “Build an eye!” program? In some sense the answer must be yes, but not at the cellular level. The cells themselves are just following a script of “if this happens, then do that!” cues that evolution wrote in the DNA [825, 1094]. If we were to ask a cell, “What are you doing?,” it could answer, but if we asked, “Where are you headed?,” it could not.

Epilogue

Fly genetics in the 1900s succeeded in deciphering the logic of disc development. Its vaunted offspring – the ﬁeld of ﬂy genomics – is faster and sexier but no more powerful in its ability to solve the remaining riddles of circuitry and control. Curt Stern warned us about this irony in his essay, “The journey, not the goal”: One of the fundamental aspects of science is its lack of purpose. . . . Science, during the last one hundred or more years, has been in the dangerous position of a successful poet who started by composing songs of joy and sorrow to lighten the burden of his own soul only to find that they became best-sellers. . . . Science has become a profession. . . . The later comers [have] forgotten the beginnings of the highway. Dreamy followers of crooked paths [were] their predecessors. . . . We should encourage anew the roaming after knowledge for the sake of the joyful adventure. [4093]

Industrialization and commercialization notwithstanding, the Fly World still offers many mysteries for aimless explorers with curiosity alone to ﬁll their sails. Before launching the ﬁeld of ﬂy genetics, Thomas Hunt Morgan’s passion was embryology [72, 3903]. In the

256

preface to his 1934 book, Embryology and Genetics, Tom waxed lyrical about the promise of developmental genetics as a burgeoning hybrid ﬁeld [2949]: Since 1900, when the discovery of Mendel’s work became known, one of the most amazing developments in the whole history of biology has taken place. The fundamental laws of heredity are now known, and since it is through the egg that the hereditary properties of the individual are carried on from generation to generation, the importance of an understanding of development to supplement the knowledge of the laws of heredity is apparent, and the interlocking of these two experimental branches of biology has become a subject of absorbing interest. . . . That much remains to be done will be only too obvious, but with the openings furnished by the experimental investigation of heredity and embryology there is promise that a great deal more is within our reach.

This hybrid has now borne fruit, and the fruit ﬂy adorns the most glorious branch. Who could ever have guessed that this little “gnat” held such rich insights in its shimmering golden ﬂeece?

APPENDIX ONE

Glossary of Protein Domains

All protein domains that were mentioned in the text or tables are inventoried below. For further information, consult PROSITE (www.expasy.ch/prosite) or the following reviews: domains in general [829, 1089, 3295], DNAbinding domains [3284], scaffolding domains [3299], extracellular domains [404], domain classiﬁcation [4219], domain evolution [3021], protein-protein binding [2091], protein-peptide binding [3299], receptor-ligand binding [3080], signal transduction [3783], and an inventory of ﬂy protein domains [3674]. D. melanogaster proteins vary in size from 21 a.a. (L38, a ribosomal protein) to 5201 a.a. (Kakapo, a cytoskeletal component needed for intercellular adhesion) [14]. The domains listed below vary from 4 a.a. (WRPW) to ∼270 a.a. (PAS). N.B.: Customarily, “domain” denotes a motif in proteins, while “box” refers to DNA. Thus, for example, the homeobox encodes the homeodomain. “Repeat” does not connote identity within a protein (e.g., only 6 of the 38 residues are invariant among Notch’s 36 EGF-like repeats [2210]), nor does it imply interchangeability. For instance, the LIM domains of Lim3 can replace those of Apterous in wing development but not in the CNS [3159]. Likewise, Cactus’s ankyrin repeats cannot functionally substitute for those of Notch [1053]. The speciﬁcity of repeats is epitomized by the “arm” domain: Although individual repeats within a single protein are only about 30% identical, they are highly conserved during evolution. Thus, corresponding repeats of armadillo and β-catenin (which are direct homologs) are very similar (e.g., repeat 1 of armadillo is 90% identical to repeat 1 of β-catenin). Individual repeats, once free to diverge, are now fixed in sequence, presumably reflecting some well-conserved interaction with a target protein. [3312]

“Peptide-binding” and “protein-binding” both indicate mortise-and-tenon “docking” sites [1950, 3834], but the former ﬁt ≤∼10 contiguous amino acids, while the latter recognize larger or discontinuous epitopes [1730, 2364, 4871]. Abbreviations: “a.a.” (amino acid), “a.k.a.” (also known as), “n” (variable nucleotide), “p ” (phosphorylated a.a. such as Sp ), “x” (variable a.a., with subscripts such as x 4 indicating the number of xs in a series). Standard 1- or 3-letter a.a. codes are given below. Sequences (a.a. or nucleotide) are underlined, and residues are listed in amino-carboxy or 5 -3 order. A C D E F G H I K L M N P Q R S T V W Y φ

Ala Alanine Cys Cysteine Asp Aspartic acid Glu Glutamic acid Phe Phenylalanine Gly Glycine His Histidine Ile Isoleucine Lys Lysine Leu Leucine Met Methionine Asn Asparagine Pro Proline Gln Glutamine Arg Arginine Ser Serine Thr Threonine Val Valine Trp Tryptophan Tyr Tyrosine (a hydrophobic a.a.)

257

258

14-3-3 The numbers 14, 3, and 3 refer to chromatographic (14th of 15 fractions) and electrophoretic (3rd of 4 quartiles) mobilities of a founding class of mammalian brain proteins (band #3 in the 14-3 group) [2915, 2916], although such proteins are widespread among eukaryotes and participate in various cellular processes [42--44, 561]. Each mammalian isoform is named with a Greek letter (from α to η) based on its order in an HPLC elution sequence [44, 1958], and ﬂy orthologs are named accordingly (e.g., 14-3-3δ). These proteins are ∼260 a.a. long [44] and bind Sp -containing sequences that have the consensus RSxSp xP or Rx(Y/F)xSp xP [3023, 4787]. Although 14-3-3 is not a bona ﬁde domain because it is not subsumed within larger proteins, it resembles PTB and SH2 domains insofar as it binds a cognate phosphopeptide. Monomers form 9 antiparallel α-helices [2567, 4770] and assemble into homo- or heterodimers [43, 2087]. Each 143-3 dimer is shaped like a cradle whose cavity is lined with negatively charged, highly conserved residues. The outer edges of the cavity bind (non-14-3-3) Sp -bearing guests [4787], with the cognate peptide in an extended main-chain conformation. In Drosophila, 14-3-3 isoforms serve scaffolding functions in different RTK signaling pathways [716, 2279, 4317] by binding to dRaf and Ksr [3639]. The details of how 14-3-3 dimers wrestle dRaf into one conformation vs. another are still being worked out [2957, 4116], but it is now clear that each half of the 14-3-3 dimer grips dRaf in a different way [43, 3638, 3639]. ADAM “ADAM” stands for a disintegrin and metalloprotease domain, and a synonym is “MDC” (metalloprotease-disintegrin-cysteine-rich) [346, 369]. It contains a putative integrin-binding “disintegrin” moiety and a protease that is typically zinc dependent [346, 369, 4721, 4722]. Some ADAM family members (e.g., Kuzbanian [3479] and TACE [491, 2995]) are “sheddases” that snip extracellular parts from other surface proteins on the same cell [3469, 4599], although the protease portions of others are nonfunctional [4721]. Other ADAM proteins may degrade extracellular matrix or assist in cell migration or adhesion [346, 3469]. ankyrin Named for a motif tandemly repeated 22 times in human erythrocyte Ankyrin, this ∼33-a.a. protein-binding domain (a.k.a. “cdc10/SW16”) [2624, 2841] is found in the Notch [1053, 2299, 2746] and IκB families [366, 2208, 3123, 4476], in various transcription factors [235, 403], in some yeast cell-cycle genes [2624], and in the putative ion channel (NompC, 29 repeats) that transduces touch stimuli in ﬂy bristles [4527]. Its secondary structure is an L-shaped “girder” that stacks stably when tandemly repeated [1564] – a fact that explains why virtually all pro-

APPENDIX ONE. GLOSSARY OF PROTEIN DOMAINS

teins that contain this domain have ≥4 consecutive copies [235, 403]. Some proteins use these modules to dimerize with other ankyrin-domain partners [1269, 2747], whereas others use them to dock with RH (Rel homology) domains of Rel-family transcription factors [168, 366, 2208, 3123] or other ankyrin-deﬁcient partners [1870]. Surface residues dictate binding speciﬁcity [168]. Ankyrin itself “anchors” (hence the name) integral membrane proteins to the cytoskeleton and has one conserved (and 14 mostly conserved) residues in its repeats.

arm First identiﬁed in the ﬂy gene armadillo (arm), where it is tandemly repeated 12 times (with one interruption) [892, 3320, 3598], the arm domain is a 42-a.a. proteinbinding module [3146, 3312]. In β-catenin (Arm’s vertebrate ortholog [3306]), each repeat forms an open triangle of 3 α-helices, and the (slightly nonplanar) triangles stack atop each other to build a cylindrical right-handed su˚ long and 35 A ˚ wide) [1593, 1936]. The (conperhelix (110 A ˚ groove that tinuously wound) superhelix has a 95-A could ﬁt a stretch of 25--30 a.a. (like a hot dog in a bun). Indeed, the groove is strongly positively charged and the binding partners of β-catenin (cadherins, Tcffamily transcription factors, APC) all have an excess of negative charges in their binding domains, so the partners likely adhere via charge complementarity [1936]. When the groove is not occupied by them, it may be ﬁlled by the acidic C-terminal tail of Arm/β-catenin itself [892]. The core of the superhelix is hydrophobic, which explains why no proteins have ≤6 tandem arm repeats (i.e., too few hydrophobic contacts for stability). Included in this family are importins α (8 repeats) and β (11 repeats) [2743], which escort NLS-bearing proteins into the nucleus [1568, 1569], and arm repeats may enable proteins to enter the nucleus (sans a NLS tag) [1180]. bHLH The ∼60-a.a. “basic Helix-Loop-Helix” domain [2566] typically resides near a protein’s N-terminus [899, 3375], although Atonal [2040] and Tap [576, 577, 1402] are exceptions. The basic part binds DNA via positively charged amino acids, while the amphipathic helices (separated by a loop of variable length) mediate dimerization [1152, 2634, 3016] or, when not bound to DNA, tetramerization [245]. Dimers have dyad symmetry and a scissors shape (Fig. 2.5), with the “blades” straddling DNA in its major groove [1152] and adopting an induced ﬁt in some cases [1221]. Most bHLH dimers bind the consensus sequence CAnnTG [348, 3017], but dimers from the E(spl) Complex bind CACnAG [3171], and bHLH-PAS proteins bind ACGTG or GCGTG [899]. The identity of the variable (“n”) nucleotides depends on the dimer [348], as do the preferences for ﬂanking nucleotides [1245], with each

APPENDIX ONE. GLOSSARY OF PROTEIN DOMAINS

member of a dimer dictating its own “ideal” half-site [899], although homodimers may bind asymmetric sites [348]. The basic part may also mediate interactions that do not involve DNA binding [764]. In bHLH subfamilies that have an auxiliary dimerization domain [135] – leucine zipper [245, 3375] or PAS [899, 900] – the other domain typically resides just C-terminal to the bHLH motif. Advantages of having two separate dimerization domains in the same protein include added afﬁnity, combinatorial selectivity, or both [1221, 4857]. Such versatility is epitomized by the bHLH-PAS proteins that implement the circadian clock (see PAS below).

BTB This ∼120-a.a. protein-binding domain is found in the ﬂy proteins Broad-Complex, Tramtrack, and Bric a` brac (hence the name; a.k.a. “POZ”) [211, 1516, 3636, 4891]. Located usually at the N-terminus, the BTB motif reduces the DNA-binding ability of zinc ﬁngers within the same protein, presumably because the oligomerization that it mediates obscures those DNA-binding surfaces [211]. (Analogous “self-defeatism” afﬂicts LIM-HD proteins [929].) In other cases, however, oligomerization facilitates DNA binding over a region large enough to bend DNA and exclude nucleosomes [2147]. Some transcription factors use their BTB domain to recruit a synergistic co-repressor [1036]. For example, the zinc-ﬁnger factor Tramtrack uses its BTB motif to recruit the co-repressor dCtBP [4596]. Tramtrack can form heterodimers, but most BTB proteins only homodimerize. COE Named for the Drosophila gene collier (a.k.a. knot) and the mammalian transcription factor Olfactory-1 (a.k.a. Early B-cell factor) [909], this ∼210-a.a. DNA-binding domain can mediate dimerization and transcriptional activation [1679]. COE family proteins also contain a C-terminal HLH domain [192] and resemble bHLH transcription factors. Collier helps specify muscle cell fate in embryos [910] and intervein identity in the wing [1840, 2894, 3077, 4479]. cut In addition to its homeodomain, the ﬂy gene cut has 3 copies (sharing 55--68% a.a. identity) of a 60-a.a. motif termed the “cut repeat” [370]. In a mammalian cut counterpart, one repeat binds DNA independently of the homeodomain [2528], but none of the ﬂy’s repeats appears capable of doing so [87]. Rather, they may modulate the strength or sequence speciﬁcity of DNA binding by the homeodomain. DEF DEF is a domain that recruits MAP kinase (a.k.a. ERK). “DEF” stands for “Docking site for ERK, FxFP” [2007]. The DEF peptide (FxFP) has two bulky hydrophobic (F) residues that are thought to ﬁt into a pocket

259

on MAP kinase. After MAP kinase docks with its DEFbearing target (e.g., Yan), it phosphorylates S or T substrates near the DEF site.

DEJL Like DEF, DEJL (a.k.a. “D domain” [2601, 4800, 4801]) is a binding site for MAP kinase, but it can also bind JNK (Jun N-terminal Kinase). “DEJL” stands for “Docking site for ERK and JNK, LxL.” It consists of an (L/I)x(L/I) tripeptide that has a cluster of basic residues on its Nterminal side [2007]. EGF-like Named for Epidermal Growth Factor (a 53a.a. soluble fragment cleaved from a ∼1200-a.a. transmembrane precursor), this ∼40--50 a.a. protein-binding module has 6 conserved cysteines that pair to form 3 disulphide bonds. The 1st Cys binds to the 3rd, the 2nd to the 4th, and the 5th to the 6th, thus weaving a knot [970, 3521] whose loops dictate binding speciﬁcity. Notch’s consensus is Cx4 Cx5 Cx8 CxCx8 Cx6 [4611]. EGF-like repeats are found in extracellular matrix proteins or extracellular moieties of membrane-spanning proteins (ligands or receptors). Some EGF-like domains use 7 oxygen atoms from their side groups to form a cage that harbors Ca2+ . A canonical Ca2+ -binding sequence is seen in at least 6 of Notch’s 36 repeats (including the two that bind Delta) and in one of Delta’s 9 repeats [1707, 3521, 3544], and NotchDelta binding is (for this reason?) Ca2+ -dependent [1204]. The world record for number of repeats in a single protein goes to Dumpy, with 308 EGF-like motifs in an extracellular domain that stretches to nearly 1 micron in length [4668]! EH Named for “Eps15 Homology,” this ∼66-a.a. peptide-binding domain has 4 invariant and 21 mostly conserved residues [4740]. Human Eps15, which has 3 tandem repeats, is a substrate for the EGF receptor and other tyrosine kinases. EH domains use a hydrophobic pocket (braced by 4 α-helices) to bind a tripeptide “NPF” that is found in Numb near its C-terminus (but in few other ﬂy proteins) [979, 3725]. Various EH- and NPFbearing proteins aggregate to control clathrin-mediated endocytosis [739, 2699, 2756, 3596, 3856]. An EH partner of Eps15 has been implicated in the EGFR signaling pathway in ﬂies [594]. Ets Named for the ets (E twenty-six) vertebrate proto-oncogene [3467], this ∼85-a.a. domain functions in both DNA binding and protein-protein docking [2135, 4559]. It binds as a monomer to 10 b.p. sequences with a GGA core. Its C-terminal half is highly basic, whereas its N-terminal half has 3 conserved Trps at ∼20-a.a. intervals. The ﬁrst Trp is imbedded in a helix, while the third resides in a NLS motif.

260

F The F domain is a ∼40-a.a. protein-binding sequence ﬁrst identiﬁed in cyclin F [169]. It links proteins to ubiquitinating enzymes and thus can act as a destruction motif [169]. F-domain proteins that contain other kinds of protein-binding sites (e.g., Slimb, which has WD repeats [2060, 4279]) are evidently hubs for assembling ubiquitination complexes [3500, 4031, 4701]. GUK The ∼160-a.a. guanylate kinase homologous (“GUK”) domain is found near the C-terminus of MAGUK (membrane-associated GUK) proteins [533, 3419]. Given their enzyme homology, it is surprising that GUK domains appear catalytically inert [1062]. Instead, they tend to mediate protein-protein interactions, possibly via a leucine zipper subdomain [4237]. The role of this domain remains obscure, but at least one of its duties is to steer host proteins to neuromuscular synapses [4300]. MAGUK proteins typically form scaffolds for assembling complexes of membrane receptors [843, 1001, 1062, 4371] and ion channels [83, 896, 4811]. Some of those complexes are crucial for growth control [533, 4561]. At least one MAGUK (CASK) leaves its membrane perch (when a signal is received) and goes to the nucleus, where it functions as a co-activator [450, 1920]. A 28-a.a. MSR (MAGUK-speciﬁc repeat) motif (of unknown function) is found in the MAGUK family [3419]. HLH The Helix-Loop-Helix protein-binding motif usually adjoins a stretch of basic residues that mediate DNA binding (see bHLH above). Alternatively, it may adjoin a DNA-binding domain of the COE class [909]. Proteins that contain a HLH domain alone cannot bind DNA. Some of them (e.g., Extramacrochaetae [1156, 1388]) sequester bHLH partners in inactive heterodimers [213, 286, 3083]. This “kidnapping” strategy is also used by transcription factors in the LIM-HD family (Beadex disables Ap via Chip [2854, 3908]) and the POU-HD family (I-POU disables Cf1a [4380]). Other types of antagonists interfere with bHLH function by protein-protein binding outside the bHLH domain [2046]. HMG This ∼80-a.a. DNA-binding (and DNA-bending) motif occurs in certain High Mobility Group (nonhistone chromatin) proteins [316] and in various other animal, plant, and yeast proteins [2035, 2402, 2415]. In one HMG subfamily, there are multiple HMG motifs per protein and DNA is recognized with little or no sequence speciﬁcity, whereas in the other subfamily (including Sox-type HMG domains [424]) there is a single HMG motif and DNA binding is sequence speciﬁc [810, 1629], although much of the speciﬁcity may depend on DNAbinding partners [2122]. HMG domains are L shaped, with two α-helices in one arm and one α-helix in the other.

APPENDIX ONE. GLOSSARY OF PROTEIN DOMAINS

They bend DNA [253, 4299, 4717] by inserting hydrophobic side chains (along DNA’s minor groove) into the stack of bases [317, 1480, 2605]. Such insertions (plus a.a.-nucleotide hydrogen bonds) unwind the double helix [1481, 3008]. The “oregami” folds thus created in promoter DNA facilitate construction of an “enhanceosome” for initiating transcription [879, 1188, 4274] (e.g., the Pan-Arm complex [849]).

homeo The ∼60-a.a. homeodomain (HD) binds DNA [1417, 1422, 1858] or, in some cases, RNA [710, 3541, 3606] or protein [2207, 3402]. It is encoded by the strongly conserved ∼180 b.p. homeobox (HB) [1420] that is named for its prevalence among homeotic genes [1418] (cf. Ch. 8). HB genes appear to be “master control genes” for body patterning [1416] . The term “Hox” (homeobox) is conventionally only used for homeobox genes within the clusters that govern body segment identities (i.e., BX-C and ANTC in ﬂies [1223] ). The HD consists of 3 α-helices (#2--4) around a hydrophobic core, plus a ﬂexible N-terminal arm containing a fourth (#1) helix [1417, 1420, 3284, 4198]. Residues ∼30-50 within the HD (helices #2 and 3) form a helix-turnhelix (HTH) [335] that is superimposable on the HTH of prokaryotic repressors [3482] despite (1) a lack of appreciable sequence identity [1416, 3219] and (2) a different set of DNA contact sites [31, 1422, 1729, 2240, 4384]. One HD subclass called “TALE”(Three Amino Acid Loop Extension) has 3 extra a.a. between helices #1 and 2 [567, 568], whereas another has 10 extra a.a. between helices #2 and 3 [1335, 3033]. In Extradenticle, the TALE region forms a hydrophobic pocket that ﬁts a hexapeptide motif (see below) [2207, 3281, 3393]. Particular HDs bind speciﬁc nucleotide sequences [857] , as evidenced by “domain swap” experiments [2071, 4879] . They function as transcriptional activators or repressors for a variety of downstream “realizator” genes [407, 1580, 2923, 2938, 3457] . The universe of potential targets [2541, 4532] is reduced in vivo [707] (vs. in vitro [709]) due to (1) the masking of inactive genes by chromatin [655] and (2) the docking preferences of adjoining trans-acting factors in the cis-enhancer vicinity [1767, 2539]. HD proteins can bind DNA as monomers [1416], dimers [4688, 4689], or higher-order complexes [3716]. In some cases, binding is regulated by phosphorylation [300, 2011, 2677]. Many HD proteins have a second DNA-binding motif [1417] that alters the HD’s DNA afﬁnity or speciﬁcity [2098], or bends DNA [4477]. Auxiliary motifs include the cut [87] , LIM [1862] , paired [2098] , and POUspeciﬁc [3713] domains, and one of these motifs (paired) can trans-complement the HD even when detached [2873].

APPENDIX ONE. GLOSSARY OF PROTEIN DOMAINS

The central two base pairs in the ∼10 b.p. bipartite binding site encode much of the speciﬁcity [709], which makes sense because the HD’s N-terminal arm binds them, and it is also critical for speciﬁcity [1417, 2671, 4846, 4862]. Most HD proteins have NLSs that send them to the nucleus [1020, 4879] (e.g., Hth [2045]), but some (e.g., Exd [3716]) also have NESs that can divert them to the cytoplasm [2661]. DNA-binding afﬁnity [3324, 3325], DNA-sequence speciﬁcity [706, 2675, 4450], and DNA curvature [3842] can be modulated by separate cofactors [683, 2421, 2539, 2674, 4623], including Extradenticle (Exd) [156] and Homothorax (Exd’s escort for nuclear entry) [2360, 3716], both of which are also have a HD [2677]. Hox proteins capture Exd by inserting an N-terminal hexapeptide φ(Y/F)(D/P)WM(K/R) (a.k.a. YPWM [879]) into Exd’s hydrophobic TALE pocket [2207, 2944, 3281, 3393, 3716, 3842]. Exd’s chief role may be to coerce its partner into a new shape [708, 2675] that (1) alters the oligonucleotide speciﬁcity of the HD itself [2675] or (2) unmasks an activation [2535, 3391] or repressor [3715] domain elsewhere in the HD protein. The Hox-Exd tryst helps explain the astounding ability of some HD proteins to function without their HD [873, 2360]. To wit, the “jigsaw puzzle” of the HD protein and its cofactors can remain intact even with a few pieces missing. N.B.: Steric cooperativity wreaks havoc for other analyses in this book – e.g., the Cubitus interruptus complex (App. 6) and the Eya-Dac-So complex (Ch. 8).

Ig-like The immunoglobulin-like (Ig-like) domain deﬁnes a superfamily of cell-surface glycoproteins [4680]. The Ig motif folds into a “β-sandwich”: two β-sheets (3 to 4 antiparallel strands per sheet) held together by one conserved Cys-Cys disulﬁde bond and by hydrophobic contacts. The sandwich (whose outer surfaces are hydrophilic) is a platform for displaying variable groups (peptide or sugar) on its faces or at the U-turns within its sheets. Ig-domain proteins commonly mediate adhesion or recognition by dimerizing (homo- or hetero-) with counterparts on opposing membranes [1296]. The vertebrate immune system may have evolved by giving these old molecules new duties in antigen binding and intercellular communication [4680]. The notorious diversity of antibodies in vertebrates may have a counterpart in ﬂies [3793]: Dscam has 10 Ig-like domains – 3 of which (plus the transmembrane domain) can be varied (by alternative splicing) to produce 38,000 potential isoforms! leucine zipper This ∼30-a.a. dimerization domain has a leucine every 7 a.a. [2401, 2790, 3219]. The leucines align along one face of an amphipathic α-helix, and 2 such helices (separate proteins) join by crossing (at a ∼20◦

261

angle) and intertwining into a left-handed “coiled coil” [1864, 2621]. The coil is stabilized hydrophobic contacts between the leucines [1718, 1719, 3163]. The term “zipper” is thus misleading because the leucines do not interdigitate [1864, 2790]. At one end, the helices diverge into a “scissors grip” around DNA [3642, 3769, 4494]. The DNA-binding tips have a separate basic region [55, 915, 1153, 3160], hence the composite “bZip” domain [2197]. Some (“bHLH-Zip”) proteins contain basic, leucine zipper, and HLH domains [245, 1594, 3375]. Whether a bZip protein forms homoor heterodimers depends on its ability to make interhelical salt bridges with its partner [1513, 2334, 4493]. Because zippers can form higher order complexes (dimers of dimers), they can mediate binding to two separate DNA sites simultaneously, thus “tying” a DNA loop that may be instrumental in transcriptional activation [1221]. Their ability to bend DNA [3251] may facilitate the looping. Large-scale versions of leucine zippers appear to be used by transmembrane proteins (like glue) to join cell surfaces together [2323].

LIM Named for Lin11 (nematode), Isl-1 (vertebrate), and Mec-3 (nematode) proteins [160], the LIM domain has two tandem zinc ﬁngers [973, 3736]. LIM repeats themselves tend to be tandemly clustered. Unlike orthodox zinc ﬁngers, LIM domains bind proteins instead of DNA [97, 929, 972, 1862]. They can interact inter se (selectively or promiscuously, depending on developmental context) [3159, 4310] or with heterologous “LIM-binding” domains [161, 929, 3786, 4456]. In some LIM-HD proteins, the LIM domains prevent the HDs from binding DNA [929, 3735]. Apterous may be one such masochistic molecule, with Chip being its rescuer (cf. Ch. 6) [2854], but the story is not so simple [2851]. LNG This ∼31-a.a. motif is tandemly repeated 3 times in Notch, and in the nematode proteins Lin-12 and Glp-1 (hence the name) [2222]. Like EGF-like domains, the LNG domain has 6 conserved cysteines, but they are spaced differently (Cx4 Cx8 Cx3 Cx4 Cx6 C instead of Cx4 Cx5 Cx8 CxCx8 Cx6 ) [4611]. Removing these repeats causes a ligand-independent GOF phenotype [2542, 3543], so they may prevent activation of the receptor in the absence of ligand (by affecting oligomerization?) [2299]. MADS The MADS motif is named after Minichromosome maintenance-1 (yeast), Agamous (Arabidopsis), Deﬁciens A (Antirrhinum), and Serum Response Factor (mammal) [3819]. Mutations in agamous and deﬁciens cause homeotic changes in ﬂoral anatomy comparable to the cell-type (vein-intervein) switching seen with LOF defects in the ﬂy’s SRF gene blistered [2907]. Thus, animals and plants both use MADS-box genes to

262

dictate histotype [3819], but to a different degree: evolution relied on MADS boxes for the selector-gene circuits in plants [1892, 3105, 3477], but it favored homeoboxes for wiring such circuits in animals [660, 1223, 2272]. The MADS domain is ∼56 a.a. long, with 18 highly conserved residues, basic residues near the N-end and hydrophobic ones near the C-end [3907, 4392]. The N-terminus serves mainly for DNA binding, with the C-terminal part forming surfaces for protein interactions (dimerization plus accessory proteins). Dimers bind a palindromic decamer whose core 6 b.p. are typically A or T [3907], although each MADS factor binds a speciﬁc cognate target. Given these variations and the ability of MADS proteins to form heterodimers, different sites could be targeted by deploying certain subsets of MADS proteins just as bHLH proteins are mixed and matched during neurogenesis (cf. Ch. 3). However, there is no evidence that Blistered dimerizes with other partners [2907]. The 3-D structure of the SRF core bound to DNA [3323] reveals 4 levels in the dyad [4392] from the DNA outward: (1) Nterminal tails that line the minor groove, (2) antiparallel amphipathic 7-turn α-helices (one per monomer) that ride the DNA backbone along the groove and hold the dimer together with help from upper levels, (3) a β-sheet of 4 antiparallel ribbons whose hairpins touch the DNA, and (4) a cupola of two 3-turn α-helices that are outside the MADS domain.

NES “Nuclear Export Sequences” are typically ∼10-a.a. leucine-rich peptides that cause proteins to leave the nucleus [1267, 2256, 3040]. The NES-binding protein Exportin escorts outbound cargo through the nuclear pore complex [953, 4057, 4419]. An interesting case study in the tug of war between NESs and NLSs is the heterodimer of Extradenticle and Homothorax [9, 28, 302, 3716]. N.B.: Certain “cytoplasmic localization” domains can keep proteins in the cytoplasm without resorting to NESs per se [1949, 1974, 3612, 3976]. NLS “Nuclear Localization Sequences” cause proteins to enter the nucleus [1566, 2032, 3040]. These ∼5--10 a.a. (mono- or bipartite) peptides act like passwords for big proteins (≥ ∼45 kD) to traverse the nuclear pore complex [4123]. (Smaller proteins trafﬁc freely.) NLS-binding proteins (importins or transportin) dock with the NLS tag [1066, 2033], escort the cargo through the pore [1565, 1567, 3413, 3581, 4598], and then unload it [2637, 2750]. Unlike signal peptides, NLSs are not cleaved during transport. All NLSs contain clusters of hydrophilic residues, which must be superﬁcial to function. NLSs are often rich in Lys and Arg, but not all such clusters serve as NLSs [3117],

APPENDIX ONE. GLOSSARY OF PROTEIN DOMAINS

and neutral residues [2663] or phosphorylation [1682] can be crucial. Whether a putative NLS truly acts as such can only be ascertained by mutating it or by putting it onto an inert “tester” protein. No consensus sequence exists in any of the 3 classes [3039], despite earlier indications [730].

opa Consisting almost entirely of glutamines (QQQQQ ...), the length of this ∼30-a.a. motif (a.k.a. “strep” or “M”) varies widely. It is named for odd paired (opa) – a pair-rule gene that has two repeats [285]. Q is the most often repeated a.a. in eukaryotic proteins having >16 tandemly repeated residues [1067], and opa-like stretches are found at over 300 sites in the ﬂy genome. In some genes, the reading frame is shifted so that the iterated CAG or CAA codons yield pure strings of a different a.a. [4615]. The function of opa motifs is unclear. They might be transcriptional activation domains [1280, 2878, 4022], or they could be “polar zippers” (cf. leucine zippers) that mediate protein oligomerization via hydrogenbonded, antiparallel β-strands [3353, 3354]. Runaway aggregation of opa-bearing proteins was thought to trigger various neurodegenerative diseases by recruiting caspases or clogging proteasomes. For example, expressing a 20-Q transgene in the ﬂy eye does no harm, whereas a 127-Q transgene causes retinal degeneration [2169]. However, the etiology remains enigmatic [3201] . paired Named for the ﬂy gene paired [456, 4785], this ∼130a.a. domain binds DNA sequences (e.g., in the even skipped promoter) [4383]. “Pax” denotes genes that contain a paired box [1632, 1757, 3125], and most Pax proteins serve as transcription factors in organogenesis [940, 1847, 1861, 4175]. In Paired and its relatives, the paired domain is associated with a homeodomain [303, 1325, 2098, 2873, 4383], whereas in others (e.g., Paired box-neuro) it is not [400, 401]. Only the N-terminal ∼70-a.a. portion of the paired motif is essential for DNA binding by Paired [303, 608, 1325, 2873, 4383], while other members of the “Pox-Pax” family use both the N-terminal (PAI) and C-terminal (RED) subdomains [934, 2098, 2314]. The different strategies seem to hinge on a single residue in the RED subdomain [4776]. One ﬂy gene (eye gone) has been found with a RED motif (and a homeodomain) but no PAI motif [2090, 2099]. PAS Period (circadian rhythm in ﬂies), Arnt (aromatic hydrocarbon receptor nuclear translocator in mammals), and Single-minded (regulator of midline fates in ﬂy CNS) share this ∼270-a.a. domain, which contains two ∼50-a.a. direct repeats (one near each end) [901, 1710, 3044]. The PAS motif folds into a β-sheet (5 or 6 strands) ﬂanked by 2 α-helices [900]. It mediates homo- or

APPENDIX ONE. GLOSSARY OF PROTEIN DOMAINS

heterodimer formation [1935], with or without an adjoining bHLH domain [899, 900]. Dimers can also form by binding of PAS to non-PAS domains [3575], and some of the partners that are recruited by a PAS domain can affect the DNA-binding speciﬁcity of the bHLH domain [4857]. Among bHLH-PAS proteins, Tango is a ubiquitous binding partner in ﬂies [1166, 4022, 4548, 4857] (cf. Daughterless’s role in the proneural bHLH subclass [588, 905, 4435]). The PAS domain is a critical gear in the clockwork that controls circadian rhythms (e.g., [1792]), so a brief libretto seems apt here. Although details are still being worked out [2486, 4821, 4822], this “bHLH-PAS Circadian Clock” clearly uses four key proteins: Clock and Cycle contain both PAS and bHLH domains [69, 165, 3710], whereas Period has a PAS but no bHLH domain [794, 3044], and Timeless has neither [3024]. Binding of Period and Timeless hides a cytoplasmic localization motif [1425, 2459, 3575, 3720] and lets an exposed NLS steer the dimer to the nucleus [926, 4505], where it indirectly activates transcription of clock [165]. Based on what happens in mammals, Clock presumably binds Cycle, and their dimer embraces E-boxes upstream of period and timeless to stimulate transcription [3710]. Timeless vanishes (degraded) at dawn [4860], leaving Period to snuff out its own gene’s expression (and that of timeless) before being phosphorylated to death by the kinase Double-time [3654]. Whether Tango intrudes in these imbroglios is unknown [899]. Other questions concern why these events should take 24 h to complete [2395].

PDZ This ∼90-a.a. globular domain (6 β-strands and 2 to 3 α-helices [586, 1097]) binds C-terminal tails of other proteins in a groove on one face [3419] but can also homoor heterodimerize in a head-to-tail orientation by inserting a β-hairpin ﬁnger into this same groove [1192, 1849]. The archetypal PDZ subfamily binds E(S/T)x(V/I), whereas other subfamilies prefer different ligands [1062, 4019]. This versatility enables PDZ proteins to assemble complexes of signal-transducing molecules at plasma membranes [1385, 3513, 4236, 4405, 4811]. For example, Canoe and/or Pyd may tie components of Notch [4237], JNK [4236], or Ras-MAPK [2748] pathways to adherens junctions. The name PDZ (a.k.a. DHR = Discs-large homologous region) comes from three founding members of the family (Postsynaptic density protein-95, Discs-large, and Zona Occludens-1) [4744]. The PDZ backbone and groove resemble those of PTB [1730]. Quite literally, PDZ domains are the chief “nuts and bolts” of the cell [1954]. PEST Many rapidly degraded proteins (half lives ≤ a few hours) contain a ∼10--50 a.a. domain that is enriched for the amino acids P, E, S, and T (hence the

263

name) [3632]. Acidic residues tend to be clustered therein (uninterrupted by basic ones, which reside at the domain boundaries), but no consensus sequence exists. Whether PEST motifs are recognized directly by components of the ubiquitin pathway or require prior phosphorylation by kinases is not known in most cases [2572], but degradation apparently occurs in the 26S proteasome [3547]. Some proteins are quickly degraded without such motifs, and the PEST hypothesis in general remains controversial [382].

PH The ∼120-a.a “Pleckstrin Homology” domain [1474, 2487, 3876] was ﬁrst noticed in Pleckstrin (a substrate of Protein Kinase C) where it is present in two nonadjacent copies [2759]. This module is remarkable for (1) the constancy of its core scaffolding (4 β-strands in one plane, 3 β-strands in another, and an α-helix just outside this “sandwich”), despite great diversity in a.a. sequence; and (2) variability of its interstrand loops, where the ligand speciﬁcity of individual proteins often resides [1213, 2546, 3546]. This strategy (a constant core decorated with variable ligand-binding loops) resembles that of the Ig superfamily [2487]. Because most PHfamily members assist in cell signaling or play cytoskeletal roles, PH domains may be protein- or lipid-binding “matchmaker” adaptors (cf. SH2 and SH3 domains) that bring together ≥2 proteins for signaling, anchorage, or catalysis (mainly at membranes) [1819, 3783]. Whether they function directly in signal transduction (by changing shape in response to ligand binding) is unclear [2478, 3546]. POU-speciﬁc The ∼75-a.a. POU-speciﬁc domain is named for the mammalian genes Pit-1, Oct-1, and Oct2, and the nematode gene unc-86 [1832, 3713]. The domain folds into 4 interlaced α-helices with a hydrophobic core [131, 1014, 2258]. Two of the helices form a classic helix-turnhelix motif [1832]. Because POU-speciﬁc domains always adjoin a (∼60-a.a.) homeodomain (HD), the term “POU domain” usually denotes the POU-HD compound [1832, 3713]. The POU and HD components are separated by a variable (15--56 a.a.) linker. They bind DNA cooperatively in its major groove [2257, 4177], with the POU portion bending the backbone [4477] and the linker constraining the overall conﬁguration [409, 2525]. POU-HD motifs are found in various transcription factors [1160, 3649, 3712, 4569], which can bind DNA as monomers or dimers while interacting with a gaggle of cofactors [3713]. Recruitment of cofactors can depend on how the dimers are disposed along the DNA [4353]. In ﬂies, examples include Nubbin (a.k.a. Pdm-1) [336, 793, 3092, 3103] and Ventral veinless (a.k.a.

264

APPENDIX ONE. GLOSSARY OF PROTEIN DOMAINS

Drifter or Cf1a) [703, 990, 995], both of which are critical for wing development.

ﬁcity for target nucleotide sequences is thought to lie outside these oligopeptides.

PRD Consisting of HP dipeptide repeats, this motif is named for the ﬂy gene paired (prd ), which contains ∼8 imperfect HP repeats in a 21-a.a. stretch of its sequence [1306]. (N.B.: The “paired” domain is likewise named for paired but is distinct [303].) Until recently, its role was unknown [4022]. One guess was a pH-sensitive chelation of metals (for DNA binding) because histidine’s pK is close to physiological pH [2025]. In Paired itself, the PRD motif activates transcription [4785].

runt The ∼128-a.a. runt domain [2113] is named for the ﬂy gene runt [2127, 3173], which encodes a transcriptional repressor [2065]. This motif binds DNA [1256, 2112, 2113, 2366, 3331] and mediates dimerization with non-runt partners [1518, 1533, 1929, 1930, 2113]. Those partners increase the dimer’s DNA-binding afﬁnity without binding DNA themselves [1533, 2524, 4234], although runt family members can bind DNA (as monomers) without such help [3172, 4541]. The greater afﬁnity is due to a change in protein conformation [1930] and is reﬂected in stronger bending (∼60◦ ) of the DNA [1533]. The shape of the runt domain puts it in the Ig-like class of DNA-binding factors: its 10 antiparallel β-sheets form a 2-layer “β sandwich” whose two free loops insert into the major and minor grooves of DNA [294, 579, 3026].

PTB The ∼160-a.a. “Phospho-Tyrosine Binding” domain [4444, 4878] has 10 conserved residues. Its “βsandwich” structure (two β-sheets plus an α-helix) resembles PH domains so exactly (despite dissimilar a.a. sequences) that it is considered a type of PH domain [2487]. Most PTB domains bind the peptide φxNPxYp , wherein Y must be phosphorylated [3299, 4444]. Numb’s PTB domain binds (1) YIGPYp φ, where Y can be unphosphorylated [2530, 4789]; (2) an NPxx sequence (9 of which exist in Partner of Numb [2609]) that is devoid of Y; and (3) an 11-mer that also lacks Y (GFSNMSFEDFP) [765]. The afﬁnity of Numb for the latter peptide (from a natural mate) can be increased 15-fold (sic! ) by replacing MS with AA (GFSNAA ...) [4900], implying that evolution settled for a tepid tryst rather than a stable union in this case. PTB motifs bind activated receptors, including Egfr [2623, 4444, 4445] and Notch [1307, 1651], and resemble PDZ domains insofar as their ligand docks onto the edge of a β-sheet (as an antiparallel β-strand) [1035, 1730]. PxDLSx(K/H) This motif is the consensus binding site for dCtBP (Drosophila C-terminal Binding Protein) in ¨ Knirps, Kruppel, and Snail [2350, 3112, 3113]. CtBP is a short-range, context-dependent co-repressor [2180, 3112, 3113] or co-activator [3377]. CtBP may act by recruiting chromatin-remodeling complexes of the Polycomb Group [3377, 3864] (cf. App. 5). Functional variants of this binding site include PLSLV in Hairy [3430] and PVNLA in Mδ (from the E(spl)-C) [3430]. RNA-binding Several kinds of RNA-binding motifs exist [1099, 3541]. The most common is the ∼90-a.a. “RNP” (ribonucleoprotein) type (e.g., in Musashi [3035]). Its two consensus sequences are separated by ∼30 a.a.: RNP1 (8 a.a.) and RNP2 (6 a.a.) occupy the central β-strands in a 4-strand antiparallel β-sheet that packs against two α-helices [1100, 2186]. Their basic and aromatic residues are implicated in salt-bridge and ring-stacking interactions with single-stranded RNA segments. Most of the speci-

SH2 The ∼100-a.a. Src homology 2 (SH2) module binds phospho-Tyr residues that have a Yp xxφ consensus. (“Src” is from sarcoma virus.) SH2 domains are used in signal transduction by receptor tyrosine kinases (RTKs) [2910, 3298], in signal extinction [1209, 4320], and in ratcheting of substrates by non-receptor kinases [2758]. Adaptor proteins commonly use an SH2 to bind an activated RTK but an SH3 to relay the signal (cf. Fig. 6.12) [3185, 3488, 3944]. SH2s form two α-helices and two β-sheets, and the ligand ﬁts a pocket on one face in lock-and-key fashion [2365, 3215, 4526]. The 3 a.a. on the carboxy side of the Yp (xxφ) usually dictate the ﬁt [342, 3296, 3299, 4020], but ﬂanking residues can also matter [3280]. A conserved Arg at the bottom of the pocket grips the Yp phosphate. Sp or Tp would be too short to reach the Arg (hence SH2’s fetish for Yp ), and Lys cannot reach the Yp (ergo substitutions here are disabling) [3296]. The N- and C-ends of each SH2 converge – a feature that made it easy for evolution to insert or delete SH2s (like buttons) without disturbing nearby domains [3296]. Nevertheless, some SH2s are conﬁgured to nudge catalytic domains upon peptide binding [1200, 4535]. Aside from SH2 and PTB, another Tyr-binding domain probably exists [1575] because YYND is so rigidly conserved in the Shc family [2389, 2623]. SH3 The ∼50--75 a.a. Src homology 3 (SH3) peptidedocking motif typically assigns proteins to certain sites within the cell [208, 3019, 3298], although the SH3 domain of Src itself also acts intramolecularly [1200]. Unlike SH2 domains, which bind Yp , SH3 domains bind Pro-rich peptides (consensus = PxxPx), which are not modiﬁed during signaling [3299]. Hence, SH3 domains are often found (sans SH2) in adaptors not afﬁliated with

APPENDIX ONE. GLOSSARY OF PROTEIN DOMAINS

phosphorylation cascades [2777, 3676] (e.g., MAGUKs [1062, 3419]). The ligand’s variable and ﬂanking residues dictate afﬁnity for certain SH3 proteins [3298]. The orientation of the prolines is irrelevant for binding [1210, 2552, 2757] because the pocket holds each Pro by its nitrogen [3108], not by a lock-and-key ﬁt. SH3 sequences form β-barrels (5-8 β-strands), with a hydrophobic ligand-binding pocket [3298, 4828] on the side opposite the N- and C-tips. Because the tips meet, SH3 domains have the same “button” modularity as SH2 domains [3298]. Yeast have SH3s but not SH2s, implying that SH3 evolved before SH2 [2365, 3296]. SH2s probably arose with RTK signaling in metazoans.

signal Signal sequences (a.k.a. leader peptides [172, 3510]) cause proteins to be (1) inserted in the plasma or organelle membranes [90] or (2) secreted [59, 1844, 3209, 4501, 4874]. They reside at the protein’s N-terminus and have a ∼12-a.a. hydrophobic core with a relatively nonconserved sequence [3339, 3510]. The core is preceded by ∼1-10 a.a. and followed (C-terminally) by ∼6 a.a., at which point some proteins are marked for cleavage by a signal peptidase [4500]. Sox “Sox” denotes an “SRY box” subgroup of HMG (∼80 a.a.) sequence-speciﬁc DNA-binding domains distinguished by ≥50% sequence identity with the HMG domain of the founding Sox member [2415, 3360] – the human gene SRY (Sex-determining Region on the Y ) [3967]. All known Sox genes have only one HMG motif per gene, and most lack introns. Sox proteins bend DNA into an acute angle upon binding in the minor groove [1220] (cf. POU-speciﬁc domains [4477]). By such bending, it can enable nearby transcription factors to contact each other (or the basal transcription machinery) [317, 1480, 1629, 3360]. Besides this architectural role, some Sox-domain proteins (e.g., Dichaete) directly activate transcription [2635]. TEA This ∼68-a.a. DNA-binding domain is named after SV40 transcriptional enhancer factor TEF-1, yeast TEC1, and the product of the Aspergillus abaA regulatory gene [566]. It is predicted to have 3 helices. Whatever its exact structure, the conﬁguration must have been honed to perfection long ago by evolution because the TEA domain of Drosophila’s Scalloped protein is 98% identical (70/72 a.a.) to the TEA domain of its human TEF-1 ortholog [623]. Consistent with its mammalian TEA-toting cousins [2010], Scalloped cooperatively binds tandemly arranged 9 b.p. cognate sequences [1686]. It requires a tissue-speciﬁc co-activator (Vestigial) to stimulate transcription of target genes [1686, 3291, 3936], and there are hints of a co-repressor in humans [727, 728].

265

WD Named for Trp (W) and Asp (D), this ∼40-a.a. motif (a.k.a. “WD-40”) typically ends in the dipeptide WD [3072, 4449]. Each repeat forms a β-sheet made of 3--4 antiparallel β-strands [3072, 4012]. Tandem copies (4--8 repeats/protein) can form a “β propeller” whose blades radiate symmetrically from a central core [3018, 4012, 4528]. WD-repeat proteins fall into ≥6 subfamilies [1006] and serve diverse regulatory (vs. enzymatic) functions, including signal transduction, RNA processing, gene regulation (e.g., Groucho [1243]), vesicular trafﬁc, cytoskeletal assembly, and cell-cycle control [3072, 4004]. Many of these functions involve assembly of multiprotein complexes (e.g., Extra sex combs [1660, 3102, 3761, 3939, 4313]), so the main role of the repeats may be to provide docking interfaces [2289, 3072]. WRPW This tetrapeptide is found at the C-terminus of the bHLH transcription factors Hairy, Deadpan, and 7 proteins encoded by the E(spl)-C [331, 1017, 2274, 3697, 3759]. Like bait for a ﬁsh, WRPW serves to recruit Groucho – a ubiquitous transcriptional co-repressor [1243, 1244, 2064, 3278]. Groucho also binds (1) WRPY at the C-termini of proteins that have a Runt Homology domain [1243, 2496], (2) FRPW in Huckebein [1526], and (3) FKPY in Brinker (Dpp pathway) [4866], indicating a consensus of “aromaticbasic-Proline-aromatic” [4866]. zinc ﬁnger In this motif, Zn2+ ions pin together ≥4 Cys or His (or rarely Asp or Glu) residues [296, 841, 2265, 3578]. Among the ∼10 types of ﬁngers, the most common is the “Cys2 His2 ” DNA-binding module [296, 2878], which forms tandem arrays. Its ∼25-a.a. ﬁnger has a Cx2–4 Cx12 Hx3–5 H consensus sequence, wherein the central x12 “ﬁnger tip” is stabilized by hydrophobic interactions between (usually) Phe and Leu (= x3 Fx5 Lx2 ). The Cys2 side of the ﬁnger is a β-sheet (2 antiparallel strands), whereas the His2 side is an α-helix that ﬁts into DNA’s major groove [4711]. (A variant type of zinc module binds in the minor groove [4883].) In each helix, 3 residues bind ∼3 nucleotides. Adjacent ﬁngers (joined by ∼8 a.a. ﬂexible links) function nearly independently (like modular “reading heads”) [296, 1187, 2265]. Some zinc ﬁngers read the same CAnnTG (E box) sequence as bHLH dimers, and co-expressing these different tribes of transcription factors in the same cells creates odd phenotypes [1334]. The “LIM” type of zinc ﬁnger [160, 3736] is used in protein-protein interactions [97, 3786] rather than for DNA binding [929, 973] (see LIM and homeo domains above). The “RING” type of ﬁnger [1295] can act as a ubiquitin ligase in some cases [2068, 2387, 2603].

APPENDIX TWO

Inventory of Models, Mysteries, Devices, and Epiphanies

Listed below are all the major models (a.k.a. hypotheses, metaphors, scenarios) and mysteries (a.k.a. enigmas, riddles, paradoxes) discussed in the text, with deﬁnitions in parentheses. Also listed are devices (a.k.a. gadgets, tricks) and epiphanies (a.k.a. principles, rules). Asterisks indicate names that were coined for convenience (cf. [1805, 1807]). Having a taxonomy of concepts – even a silly taxonomy – is a useful heuristic for thinking [1873, 2871, 3259]. Within each category, concepts are listed alphabetically. Numbers in bold are pages where the ideas are diagramed.

Models

Pages

Activator-Inhibitor Model (activation is local while inhibition occurs at longer range) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 Angular Values Conjecture (segment polarity genes encode an angular coordinate)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Ap-Chip Tetramer Model (a transcription factor is built like a jigsaw puzzle). . . 160, 162 Arc Scenario (Dpp and Wg are constrained to travel in arc-shaped paths)* . . . . 104, 126 Basitarsal Elaboration Scenario (bristle patterns evolved as modules)* . . . . . . . . . . . 63, 64 Battery Dichotomy Hypothesis (larvae and adults use different sets of genes)* . . . . . . 87 Bias Model (Notch output is modulated by ligand input)* . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Binary Code Conjecture (regional identities are speciﬁed by binary codes)* . . . . . . . . . 85 Blinker Model (Notch output is modulated downstream of the ligand)* . . . . . . . . . . . . . . 9 Border Guard Model (segregation is achieved by preventing intermixing at the border)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149, 150 Boundary Model I and II (regional interfaces are used as coordinate axes). . . . . 102, 104 Cabaret Metaphor (transient states rely on intercellular signaling)* . . . . . . . . . . . . . . . . . 90 Cascade Model (histotypic genes are regulated through a chain of command)* . . . . 244 Catalysis Model (Notch plays an indirect role in transcription regulation)* . . . . . . . . 8, 13 Climbing Scenario (Dpp output tends to increase with time)* . . . . . . . . . . . . . . . . . . . . . 154 Clock and Wavefront Model (cells change state cyclically until hit by a wave). . . . . . . 304 Cloud Scenario (Dpp and Wg diffuse randomly)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104, 126 Coding Model (Numb plays an abstract role in specifying bristle-cell identity)* . . . . . . 7 Combinatorial Cascade Model (fates are induced sequentially by ≥2 cell contacts)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212, 214

266

APPENDIX TWO. MODELS, MYSTERIES, DEVICES, AND EPIPHANIES

Contest Model (SOP selection relies on Dl-N competition)* . . . . . . . . . . . . . . . . . . . . . . . . . 46 Crystallization Model (ommatidial cells adopt fates via contact with a template). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Cyclic Amnesia Scenario (homeosis is due to memory loss)* . . . . . . . . . . . . . . . . . . . . . . . . 85 Cyclops Scenario (signaling complexes can be “gated” by microtubule binding)*. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Differential Threshold Model (bristles ≈ hills of various heights in a rising or falling tide)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Diffusible Activator Model (SOP selection relies on accretion of inducer)* . . . . . . . . . . . 46 Distal Organizer Scenario (the future tip of the leg is the origin for all radial coordinates)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104, 129 Double-Gradient Models (two opposing gradients serve as dual inputs). . . . . . . . . . . . 124 Dpp Gradient Model (Dpp is a morphogen that assigns cell fates across the disc). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140, 142 Dpp-Wg Growth Potential Model (growth is dependent on both Dpp and Wg)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 D/V Compartment Hypothesis (the eye has bona ﬁde D and V compartments)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202, 206 Either Duo Hypothesis (either of two pairs of factors dictates antennal identity)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250, 252 Filter Model (bristle initiation relies on competence to varied signals)* . . . . . . . . . . 34, 35 Force Field Model (bristle initiation relies on a web of physical forces)* . . . . . . . . . . 33, 34 Gating Model (bristle-cell identity relies on a series of decisions as in a maze)* . . . . . . 10 Gradient of Developmental Capacity (GDC) Model (discs regenerate via gradients). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93, 94 Handcuff Scenario (Cos2 binds Ci to microtubules and thereby muzzles it)* . . . . . . . 286 Hedgehog Gradient Model (Hedgehog is a short-range morphogen). . . . . . . . . . . 138, 142 Hh-Dpp-Wg Model (1 short-range and 2 long-range morphogens are used)* . . 104, 115 Hopeful Monster Hypothesis (drastic mutations facilitate evolution). . . . . . . . . . . . . . . 237 Hox Code Model (metamere states are encoded by combinations of Hox genes). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 Hox Competition Model (Hox proteins compete for DNA-binding sites). . . . . . . . . . . . 247 Hox Isoforms Conjecture (metamere identities rely on different splicing isoforms)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Hox Threshold Conjecture (different amounts of Hox protein dictate different fates)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Hybrid PC-Boundary Model (polar coordinates cooperate with boundaries)* . 101, 102 Inhibitory Field and Wavefront Model (these 2 variables create a hexagonal lattice)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224, 226 Instructive-Permissive Model (Dpp and Wg jointly dictate wing bristle identities)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Intercalation Scenario (growth is stimulated by xenotopic contact)* . . . . . . . . . . . . 95, 154 Jacob’s Ladder Model (cross-vein positions are dictated by physical forces)* . . . . . . . 189 Lateral Inhibition Model (SOP selection occurs via a winner-take-all strategy). . . . 46, 49 Local Depletion Model (bristles are spaced apart because SOPs consume an inducer)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 MF-pushing Model I (Hh and Dpp act in a loop that drives the MF)* . . . . . . . . . . . . . . . 229 MF-pushing Model II (Hh and Dpp act with agent X in a loop that drives the MF)*. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 Minotaur Scenario (the “crawling” of a boundary is self-limiting)* . . . . . . . . . . . . . . . . . 122

267

268

APPENDIX TWO. MODELS, MYSTERIES, DEVICES, AND EPIPHANIES

Mutual Activation Model (ac turns on sc and vice versa)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Mutual Inhibition Model (SOP selection occurs via egalitarian competition). . . . . 46, 50 Mutual P-D Antagonism Model (proximal and distal domains compete)* . . . . . . . . . . 134 Nuclear Notch Model (Notch regulates transcription directly)* . . . . . . . . . . . . . . . . . . . . 8, 12 Obey Your Mother! Model (bristle-cell fates are dictated by inherited instructions)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5, 6 Omb Memory Hypothesis (the omb-on state is inherited after a certain stage)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Ommatidial Lineage Hypothesis (ommatidia develop as clones)* . . . . . . . . . . . . . . . . . . 202 Open for Business Model (more of the BX-C is accessible in posterior parasegments). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 Paravein Hypothesis (ectopic veins emerge atavistically between extant ones)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 Polar Coordinate (PC) Model (positions are speciﬁed by polar coordinates). . . . . . 93, 95 Positional Information (PI) Hypothesis (cell fates are dictated by coordinate systems). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79, 82 Posterior Prevalence Model (posterior Hox genes nullify the effects of anterior ones). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Predestined SOP Model (SOP selection is based on prepatterned biases)* . . . . . . . . . . . 45 Prepattern Hypothesis (new states are spatially constrained by prior states). . . . . . 33, 82 Raft Scenario (Hh is transported by lipid rafts)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Ratchet Model (BX-C genes enable segments to rise above a ground state)* . . . . . . . . 243 Ratchet Scenario (cells acquire states in an irreversible sequence)* . . . . . . . . . . . . . . . . 155 Rho-Vn Booster Model (wing veins are initiated by both Rho and Vn)* . . . . . . . . . . . . . 183 Rock Concert Scenario (cells become deafer as morphogen volume rises)* . . . . . . . . 147 Scalar Model (ommatidial polarity arises via a standard gradient mechanism). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209, 210 Selector Afﬁnity Model (cell assortment relies on compartment identity). . . . . . 148, 150 Selector Gene Hypothesis (regional identities are encoded by binary gene states). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Short-range Inducer Model (a third state is induced at a two-state boundary). . . . . . 107 Signal Relay Scenario (fates are assigned by a series of inductions). . . . . . . . . . . . . . . . . 139 Site-speciﬁc Enhancer Model (bristle initiation relies on unique cis-enhancers)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40, 41 Spreading Model (bristle patterning relies on diffusible inducers). . . . . . . . . . . . . . . . . . . 39 Stick-and-Straighten Scenario (cells align via polarized adhesion)* . . . . . . . . . . . . . . . . 174 Stop the Clock! Model (cells stop changing their state when they receive a signal)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214, 217 Subgene Hypothesis (heteroallelic phenotypes imply AS-C subdivisions).. . . . . . . . . . . 38 Threshold Model (Hairless titrates Su(H) and the remaining one dictates cell fate)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22, 23 Tiger-by-the-Tail Scenario (Hh is passed from cell to cell by its cholesterol adduct)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Time Window Model (SOPs can only “percolate” during a limited period)* . . . . . . . 70, 73 Tkv Barrier Hypothesis (dense receptors block morphogen diffusion)*. . . . . . . . . . . . . 143 Turing’s Model (patterning depends on diffusible reactants). . . . . . . . . . . . . . . . . . . . . . . . . 33 Use-as-needed Scenario (BX-C genes enable regions to deviate from a ground state)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 Vector Model (ommatidial polarity arises via individually polarized cells). . . . . . 210, 211

APPENDIX TWO. MODELS, MYSTERIES, DEVICES, AND EPIPHANIES

Mysteries Abruptex Paradox (what causes these odd Notch mutations?)* . . . . . . . . . . . . . . . . . . . . . . 52 Athena Enigma (how do stripes of cells arise so neatly ab initio?)* . . . . . . . . . . . . . . . . . 175 Autoinhibition Paradox (how do cells avoid repression by their own inhibitor?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Bristle Coding Enigma (how are bristle positions encoded?)* . . . . . . . . . . . . . . . . . . . . . . . . 37 Bristle Plotting Puzzle (how are bristles patterned?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Cell Competition Conundrum (why do sluggish cells end up near boundaries?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Collective Amnesia Conundrum (how do clusters of cells switch fates?)* . . . . . . . . . . . . 85 Dorsal Remnant Mystery (how does dorsal leg tissue robustly resist insults?)* . . 97, 100 Ends-Before-Middle Riddle (why do leg discs assign proximal and distal fates early?)*, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Enhanceosome Puzzle (how do transcription factors sterically execute Boolean logic?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 Euclidean Ruler Problem (how do cells align themselves?)* . . . . . . . . . . . . . . . . . . . . . . . . 173 Fickle Bristle Mystery (why do bristles seek one or the other end of a facet edge?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229, 230 Fickle Sensilla Mystery (why do sensilla seek one or the other end of a leg segment?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130, 131 Field Effect Mystery (why are the effects of some mutations nonautonomous?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Fragmented Stripe Dilemma (why are dpp-on stripe enhancers “atomized”?)* . . . . . 129 Gaps Mystery (why don’t compartment lines coincide with symmetry axes?)* . . . . 97, 98 Gearing Riddle (how do adjacent ommatidia rotate relative to one another?)* . . . . . 304 Growth Cessation Mystery (how do organs “know” when to stop growing?)* . . . . . . . . 92 Homeobox Homunculus Mystery (why are Hox genes colinear with the body?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 Homeosis Riddle (how do genes transform the identities of body parts?)* . . . . . . . . . . 237 Lattice Riddle (how is the hexagonal lattice of ommatidia created?)* . . . . . . . . . . . . . . . 224 Leg Stump Riddle (why are legs truncated by dpp LOF but not by wg LOF ?)* . . . . . . . . . . 129 MC vs. mC Paradox (why do MC and mC patterns differ if their PNCs are the same?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Memory Riddle (how do cells sustain their states of determination?)* . . . . . . . . . . . . . . 248 Nonequivalence Riddle (why do some mutations affect bristles in a spatially heterogeneous way?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 One Disc or Two? Paradox (is the eye-antenna disc a simple or compound disc?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 One Eye or Many? Riddle (what did the ancestral eye of insects and humans look like?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Photoreceptor Coding Enigma (how are photoreceptor identities encoded?)* . . . . . . 224 Planar Polarity Puzzle (how do cells orient themselves within a plane?)* . . . . . . . . . . . 304 Position-Projection Mystery (how do PNS axons ﬁnd their CNS targets?)* . . . . . . . . . . 191 Quadrant Regeneration Mystery (why does only one quadrant of the leg disc regenerate?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97, 100 Ras Speciﬁcity Riddle (how does one signaling pathway manage to evoke a variety of responses?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Registration Riddle (how does an eccentric stripe specify a symmetric pattern?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

269

270

APPENDIX TWO. MODELS, MYSTERIES, DEVICES, AND EPIPHANIES

Regulation Riddle (how do patterns regenerate and remain size invariant?)* . . . . . . . . 81 Shared Genes Riddle (how can the same genes expressed in two discs make them different?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Spacing Precision Problem (how are ommatidia positioned so regularly?)* . . . . . . . . . 228 Three Genes vs. Nine Segments Paradox (how does the BX-C specify metamere identities?)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 Triplications Mystery (why do leg outgrowths converge vs. diverge?)* . . . . . . . . . . . 98, 100 Yin Yang Paradox (how can mutually inhibited genes be co-expressed?)* . . . . . . . . . . 128

Devices (Actual or Hypothetical) bHLH-PAS Circadian Clock (a clock mechanism for daily cycles)*. . . . . . . . . . . . . . . . . . 263 Delta-Notch Flip-Flop (an ampliﬁer for small disparities in signaling)* . . . . . . . . . 46, 209 Finger Shufﬂing Trick (the use of alternative splicing to change zinc ﬁnger “addresses”) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19, 20 Heterochronic Superposition Trick (a way to create overlapping patterns without interference)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 HLH Hourglass (a clock mechanism for triggering events)* . . . . . . . . . . . . . . . . . . . . . . . . 297 Kaleidoscope Toy (evolution’s usage of Boolean logic to activate new genes)* . . . . . . 302 Kill the Stragglers! Trick (a way of robustly ensuring uniform periodicity)* . . . . . . . . . 228 Kilobase Spanner (a fastener that ties DNA into loops)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 on-then-off Licensing Trick (activation of a gene only after its upstream regulator turns off)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 POU Hourglass (a clock mechanism for counting mitoses)* . . . . . . . . . . . . . . . . . . . . . . . . 297 SOP Computer (an analog-to-digital HLH transducer)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Stratiﬁcation Device (a way to create a third state at a border between two others)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

Epiphanies AS-C Epiphany of 1995 (the AS-C inputs regional cues and outputs bristles)* . . . . . . . 44 Deaf-Speakers/Mute-Listeners Trick (inter-regional signaling is unidirectional)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Iroquois Epiphany of 1999 (the eye has bona ﬁde D and V compartments)* . . . . . . . . 203 Micromanager Epiphany of 1998 (Ubx directly controls multiple levels of target genes)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Numb Epiphany of 1994 (bristles differentiate via a binary lineage code)* . . . . . . . . . . . . 9 Principle of Nonequivalence (identical elements are encoded differently). . . . . . . . . . . 35 Proximity vs. Pedigree Rule (most fates are assigned by intercellular signaling)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Regeneration Epiphany of 1999 (regeneration does not recapitulate development)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Venn Overlap Rule (certain genes only turn on targets where their expression overlaps)* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172, 251

APPENDIX THREE

Genes That Can Alter Cell Fates Within the (5-Cell) Mechanosensory Bristle Organ

In parentheses after each gene name are the salivary gland map location and the origin of the name, except where obvious. The PHENOTYPES section only lists effects on the SOP lineage. The PROTEIN section presents available data on function, length, subcellular location, domains (see App. 1; number of repeats in parentheses), and binding partners. For further information, see FlyBase and The Interactive Fly. Abbreviations: “≈” (resembles), a.k.a. (also known as), cyt. (cytoplasm), DS (downstream in causation; “+” activated or “−” inhibited by US gene), GOF (gain of function by mutant allele or transgene construct), LOF (partial loss of function), nuc. (nucleus), PNC (proneural cluster), SOP (sensory organ precursor), US (upstream), UTR (untranslated region). Evidence for the hierarchical order of genes in a pathway (US or DS) is given from a DS perspective only. Thus, the link “a b” would be entered under gene b as “DS(+) of a” followed by epistasis evidence, but under gene a as just “US(+) of b” without the data. Likewise, “c d” would be listed for d as “DS(−) of c” with evidence, but for c as “US(−) of d” without the data. In a manner related to this class of genes, various subtypes of chemosensory neurons can be interconverted by LOF mutations in gustB [111, 3918] and abnormal chemosensory jump 6 [818] (cf. scalloped [623]). Overexpressing the proneural gene lethal at scute has no effect on the SOP lineage [3027]. Some genes were not tabulated because the basis of their phenotypes has not been studied. For example, cellular etiology is unclear for LOF alleles of seven in absentia (sina) [671] and phyllopod (phyl ) [717, 1052, 3378], which cause balding (absence of shaft and socket) or multi-

ple (2 or 3) shafts in what appears to be a single socket but may actually be multiple sockets [2680]. Sina and Phyl form a multimeric complex that promotes proteolysis of Tramtrack [2529, 4249]. Other genes were omitted because their roles are redundant (viz., the 6 other bHLH genes in the E(spl)-C [4767] and the ≥6 other Brd family genes [2382]) or seem incidental – viz., big brain (it transforms shafts to sockets when overexpressed, but only when Notch or Delta is also overexpressed [1075]), and canoe (App. 5; its LOF alleles dominantly enhance the N spl 2-shaft trait, but it has no independent effects). Inactivation of Gαi by transgenic RNAi causes unspeciﬁed fate changes [3776]. Also excluded were Suppressor 2 of zeste and Posterior sex combs, whose GOF effects appear to arise by a trivially neomorphic route [514, 3872]. The senseless gene is needed for SOP differentiation but does not seem to function in fate assignment per se within the lineage [3127] (see App. 5). LOF mutations in proteasome subunit genes cause shaft-to-socket and neuron-to-sheath transformations indirectly by increasing the concentration of activated Notch [3821]. Genes whose LOF effects on the embryo CNS suggest allegiance to this group of bristle-affecting factors include bazooka [3803, 4709], discs large [3205, 3326], inscuteable [3195] , jumeaux [729], klumpfuss [2250, 4803], miranda [3630] , partner of inscuteable [3777, 3822, 4827], partner of numb [276, 3630] , sanpodo [1128, 3263, 3985], tap [577] , etc. [609]. The same goes for glial cells missing, which is expressed in the glial cell of the bristle complex [1447] and is required for its differentiation [4438]. Additional genes were isolated in recent screens [6, 295, 555, 2903, 3471], including string, which regulates mitosis (see text). “Neural precursor” genes (not listed)

271

272

APPENDIX THREE. GENES THAT ALTER FATES OF BRISTLE CELLS

implement neural differentiation [2285] and hence are downstream of the genes tabulated here [2018]. A curious member of the precursor group is cousin of atonal (cato) [1586] – a bHLH gene outside the AS-C (53A3–5) that is expressed in SOPs after asense. Cato’s chief role appears to be in the control of neuron morphology.

asense (ase, 1B1–7, AS-C, sensory defect). PHENOTYPES: Null: twinned bristles (anterior wing margin only) that may indicate socket-to-shaft or IIb-to-IIa transformations [1079, 2039], plus other defects unrelated to fate switching. GOF: no apparent effect on intra-bristle cell fates (although extra bristles are induced) [438, 1079]. PROTEIN: Function: pan-neural transcription factor (bHLH type; 486 a.a.) [1540]. Location: nucleus [438]. Detected in SOP, IIa, and IIb, but not in the ﬁnal 5 cell types [1079]. Domains: bHLH, opa, PEST, Pro-rich region near N terminus, and acidic region centrally [1540]. Partners: binds as homodimer or Ase/Da heterodimer to “E box” DNA sequences [2039]. Bearded (Brd, 71A1–2, extra bristles) – one of 6 related genes in the Brd Complex that act alike [2382, 2383]. Pathway: same pathway as N: the extra-bristle phenotype of BrdGOF is partly suppressed by an extra dose of N+ [2500]. PHENOTYPES: Null: wild-type (i.e., no apparent defects) [2500], probably due to redundancy [2382]. GOF: switches IIa to IIb [2500]. PROTEIN: Function: unknown (81 a.a.) [2499]. May have no normal function in bristle cells, despite its ability to affect their fates when overexpressed via GOF mutations (cf. E(spl)-C). Location: unknown, although other members of the Brd family are cytoplasmic [92]. Domains: no obvious motifs except for a putative amphipathic α-helix [2383, 2499], although there are several intriguing motifs in the 3 untranslated part of Brd’s mRNA [2384--2386, 2499]. Partners: none known. Delta (Dl, 92A2, delta-shaped tips of wing veins). Pathway: US(+) of N [2542] (but see App. 5). PHENOTYPES: Null (assayed in mosaics): ∼wild-type for fates within the bristle [4859], which is puzzling given its stronger partialLOF effects. In double mutants with Ser null , Dl null switches IIa to IIb, socket to shaft, and (less often) sheath to neuron (≈ N LOF ) [4859]. Severe LOF (t.s. mutants): 4 neurons (≈ severe N LOF ) [3272, 3273]. LOF (t.s. mutants): may switch IIa to IIb (cf. its “2-neuron-2?-sheath” trait), socket to shaft, and sheath to neuron (cf. its “2-shaft-2neuron” trait) [218, 3272] (≈ numbGOF ). GOF: switches shaft to socket [2008]. PROTEIN: Function: ligand for Notch receptor (832 a.a.) [3022]. Location: transmembrane (singlepass) [2300, 4465] but also secreted under some conditions [2264, 3479]. Equal amounts in IIa vs. IIb (and shaft vs. socket

cell) [3270] argue against a “sibling rivalry” mode of fate determination here. Domains: signal peptide, EGF-like (9x), transmembrane [2300, 4465]. Partners: binds itself [2263] and N in trans (i.e., on adjacent cells) [1204, 2263, 3544] and possibly also N in cis (i.e., on the same cell) [2008].

deltex (dx, 6A, Delta-like gene on the X). Pathway: US(+) of N [2746] and US(−) of H [3027]. Suppressed by Su(dx) [1275, 1276], which encodes a ubiquitin ligase [129, 875]. PHENOTYPES: Null: unknown [580, 1053]. LOF: infrequent doubling or loss of eye bristles [1570, 4778]; main effects (thick wing veins, nicked wing margin, fused ocelli) are unrelated to bristles. GOF (≈ NGOF ): switches shaft to socket and may switch IIb to IIa (its “2-shaft-1?socket” trait may actually be “2-shaft-2-socket”) [2746]. Also switches sheath to neuron [3027]. PROTEIN: Function: unknown (737 a.a.) [580]. Although deltex overexpression alters bristle cell fates [2746], deltexLOF mutations have virtually no effect [1570, 4778], so either (1) deltex plays no role in bristle development or (2) it acts redundantly [1053]. Location: cytoplasm [580, 1053]. Domains: zinc ﬁnger (ring-H2), opa (2x), and binding site for SH3 domains – all apparently nonessential. What’s crucial is the Nterminal third, which binds Notch’s ankyrin repeats [1053, 2746]. Partners: binds N [1053, 2746] but not H [3027]. Enhancer of split (E(spl), a.k.a. m8, 96F11–14, enhances split, a NLOF allele that causes double shafts) – one of 7 bHLH genes in the E(spl)-C that act alike in many contexts [3075] (but see [871, 2548, 2549]). Pathway: DS(+) of N: lethality of E(spl)-Cnull is not rescued by NGOF [2542]. DS(+) of Su(H): Su(H)-binding sites in m8 promoter are needed for expression of m8 in PNCs (but not in SOPs) [2453]. PHENOTYPES: Null: wild-type, due to functional overlap with other E(spl)-C genes [1018, 1794, 3806]. Deletion of all 7 bHLH E(spl)-C genes (in somatic cell clones) fails to switch bristle cell fates, so E(spl)-C may have no normal role in assigning those fates [991] – a conclusion supported by the lack of any fate switches for grouchoLOF (Groucho encodes M8’s co-repressor; see App. 5). GOF: various defects including double, missing, stunted, or deformed shafts and double sockets [2384, 4256, 4318]. In contrast, overexpressing the bHLH E(spl)C genes m7, mδ, or mγ has no effect on the SOP lineage [3027]. PROTEIN: Function: transcription factor (bHLH type; 179 a.a.) [2246, 2274, 3171]. May have no normal function in bristle cells, despite its ability to affect their fates when overexpressed via GOF mutations [2384] (see text). Location: nucleus, but not in SOP [2052, 2053]. Domains: bHLH, WRPW (Groucho-binding) motif, PEST [1017, 2274]. Partners: binds Groucho [2064, 3278, 3430] and “N box” DNA

APPENDIX THREE. GENES THAT ALTER FATES OF BRISTLE CELLS

sequences (CACnAG) [3171, 4318] but not “E boxes” [4452]. Also binds (and is phosphorylated by) Casein kinase II [4395], as are M5 and M7 but not M3 or Mγ (which may help explain subgroup idiosyncrasies within the E(spl)C family).

Hairless (H, 92E12–14, bristle loss). Pathway: same pathway as numb: H LOF synergistically enhances numbLOF [4542]. Antagonistic (−) to Su(H), but epistatic relationship is unclear: double mutant of H LOF and Su(H)null has mixture of H LOF and Su(H)null traits [3827]. DS(−) of dx: double-LOF ≈ H LOF , and double-GOF ≈ H GOF [3027]. PHENOTYPES: Null: failure of SOP initiation at most sites [198]. Rare SOPs that do arise yield either a 4-socket cluster (implying a IIb-to-IIa and a shaft-to-socket switch) or a 2-sheath pair (implying a neuron-to-sheath switch and a loss of IIa) [3027]. LOF (≈ Su(H)GOF ): switches shaft to socket [198, 200, 2475], but neurons transform only partially into sheath cells [3027]. GOF (≈ Su(H)LOF ): switches socket to shaft [200, 218, 2657], IIa to IIb, and sheath to neuron [3027]. PROTEIN: Function: antagonist for Su(H). Thought to act by blocking Su(H)’s DNA-binding domain (1059 a.a.) [200, 492, 2659], H also recruits the co-repressor dCtBP [1329] ([2129] sequel). Location: nucleus [2657, 2658]. Expressed in all descendants of the SOP (glial cell is a possible exception) more strongly than in background epidermis [3027]. Domains: NLS (3x), PRD, and stretches of Ala [200, 2657]. Extremely basic overall (pI = 9.5), with 40% of its residues = Ala, Ser, or Pro [200], but also has conserved acidic sites [2695]. Partners: binds Su(H) [171, 492, 1332, 2657, 2658] and Numb [4542] but not Dx or Mam [3027]. lethal (2) giant larvae (lgl, 21A, neoplastic growth). Pathway: same pathway as numb: LOF phenotype is exacerbated when lglLOF is combined with numbLOF [3205]. PHENOTYPES: LOF (≈ numbLOF ): switches IIb to IIa [3205]. PROTEIN: Function: tumor suppressor (two splicing isoforms: 708 or 1161 a.a.) [2006, 2796] which maintains apicalbasal polarity [333, 3205, 3326, 4707], cell shape [2667], epithelial integrity [1394--1396, 4742], and compartment boundaries [35, 2975]. Location: mainly in cytoskeletal matrix at the lateral cell cortex [3205, 4134]. Domains: WD repeats [3326], a basic region, 3 different homo-oligomerization domains, a matrix recognition tripeptide, and a Ser-Thr-rich tail in the larger isoform [2014, 2247, 4133]. Partners: binds itself, nonmuscle myosin II, and ∼10 other matrix or junctional proteins [2120, 4133], probably including Discs large [3205, 3326, 4745]. mastermind (mam, 50C20–23, enlarged CNS). Pathway: Notch pathway [1000, 1514, 2353, 2394, 4780] possibly US

273

of Delta [1816]. PHENOTYPES: LOF (as deduced from constructs that act in a dominant-negative manner): 4 neurons, or switches socket to shaft (≈ NLOF ) [1816]. GOF: unknown. PROTEIN: Function: possibly a transcription factor (1596 a.a.) [1816, 4007] or co-activator for Notch [3355, 4812]. Location: nuclear in SOP, IIa, IIb, and perhaps terminal cells [305, 4007]. Domains: a basic region (with one NLS) that resembles a bZip motif [305], 2 (separated) acidic regions, 3 (clustered) stretches of alternating GlyVal, multiple opa motifs (Mam is 22% Gln!), and many homopolymeric stretches; very hydrophilic [1816, 4007, 4813]. Partners: binds Su(H) when Notch’s ankyrin repeats are also present [3355]. Does not bind H [3027]. Localizes to polytene chromosomes (mainly at actively transcribing genes), but binding may not be directly to DNA [305].

musashi (msi, 96E1–4, double shafts reminiscent of 2-sword ﬁghting style invented by samurai warrior Musashi Miyamoto). PHENOTYPES: Severe LOF: 4 sockets [3035, 3182]. LOF (≈ numbLOF ): switches IIb to IIa, and shaft to socket [3035]. GOF: unknown. PROTEIN: Function: RNA-binding protein (606 a.a.) [3035]. Msi binds tramtrack transcripts ([3035] sequel). It may act like Elav [2563, 3620, 3731, 4805] to modulate distinct RNA isoforms [2312, 2313] and thereby toggle cell fates [1857]. Location: nucleus [3035]. Enhancer-trap lacZ reporter is expressed in SOP, IIa, IIb, and all bristle cells (more in neuron and sheath than in shaft and socket cells). Domains: RNA-binding (2x), plus N-terminal (a.a. 43–127) and C-terminal (a.a. 399–577) regions that are ∼45% Ala or Gln [3035]. neuralized (neu, cf. its enhancer trap “A101,” 85C, neural hyperplasia in the embryo). Pathway: Notch pathway, but not for wing veins or margin [2387]. US(+) of N [1000, 2542]. PHENOTYPES: Null: switches IIa to IIb, socket to shaft, and sheath to neuron (≈ NLOF ) [2387, 4814]. GOF: no known effects on the lineage [2387] aside from suppression of SOPs (see App. 5). PROTEIN: Function: ubiquitin ligase (754 a.a.; see [2387] sequels) [417, 3463]. Location: cell membrane [2387, 4814]. Detectably expressed in SOPs [417, 1925], but must also function in SOP descendants based on the phenotypes of null clones [2387, 4814]. Domains: zinc (ring) ﬁnger near C terminus, NLS, opa (2x) [417, 3463]. Notch (N, 3C7, notched wing tip). Pathway: same pathway as numb: NLOF synergistically enhances numbGOF [1651]. DS(−) of numb: double-null ≈ Nnull [1651]; Nnull does not alter Numb asymmetry [1651, 3579]; fate-switchable period extends beyond numb’s [1742, 3579]. DS(+) of Dl: double mutant of NGOF and DlLOF ≈ NGOF [2542]. DS(+)

274

APPENDIX THREE. GENES THAT ALTER FATES OF BRISTLE CELLS

of dx: N GOF rescues dxLOF phenotype [2746]. DS(+) of neu: NGOF rescues neuLOF [1000, 2542]. US(+) of E(spl) [2542] and ttk [1651]; US(−) of pros [3550]. PHENOTYPES: Severe LOF: 4 neurons [1742]; presumably same in bald areas of null clones [996, 1058] and in t.s. LOF mutants [603]. LOF (≈ numbGOF ): switches socket to shaft [218, 996, 1307, 1803, 3028]. GOF (≈ numbLOF ): switches IIb to IIa [3270], shaft to socket [1307, 1651, 2627, 3270, 3543], and possibly neuron to sheath [3270]. Extreme GOF: 4 sockets [1651]. PROTEIN: Function: receptor for Delta or Serrate ligands and signal transducer (2703 a.a.) [2210, 3022, 4611]. Intracellular part goes to nucleus and serves as transcriptional co-activator with Su(H) (cf. Fig. 2.2) [2211]. Notch was also thought to assist in cell adhesion [630, 1204, 1742, 1900] and in delaying differentiation [1271], but both ideas have been discounted [112, 994, 1612]. The signiﬁcance of other isoforms is unresolved [4602]. Location: transmembrane glycoprotein (single-pass) [2210, 4611] in a subapical ring [1851]; cleaved (as precursor) in trans-Golgi at a prospective extracellular site [368]. Truncated construct (N-intra) goes to nuc. [2542, 3543, 4161], but not if Numb is added [1307]. Ubiquitous in disc epithelium, including SOPs [3270]. Domains: signal peptide, EGF-like (36x), LNG (3x), transmembrane, RAM23, ankyrin (6x), opa, PEST, and NLS (2x) [2210, 4244, 4611]. Partners: Binds itself [4601, 4823] (cf. Fig. 2.3), Big brain [4601, 4823], Delta [985, 1204, 2263, 3544, 4601], Deltex [1053, 2746], Disabled [1493], Dishevelled [151], Fringe [990, 2096, 4601], Notchless [3662], Numb [1307, 1651], Pecanex (high afﬁnity) [4601], Presenilin [3533, 3534], Serrate [2263, 3544, 4601], Su(H) [1269, 2747, 4244], and possibly Su(deltex) [129]. N also binds Wingless [4601, 4823], but not as a receptor [310, 1971, 3689], despite suspicions based on in vivo cross-talk [459, 886, 1851, 2718] and enigmatic cell culture data that is open to other interpretations [4601, 4603]. N does not bind Scabrous [2460, 4601, 4823] but associates with it in a complex [3456], so a third protein must serve as a bridge to attach them.

numb (30B, sensory defect). Pathway: US(−) of N [1651, 3579], Su(H) [3027, 4542], and ttk [1650]; US(+) of pros [3550]. PHENOTYPES: Null: 4 sockets [3579]. N.B.: The numb allele used here [3579] was considered null [4417] but may not be [552]. LOF: switches IIb to IIa, shaft to socket, and neuron to sheath [1307, 3579, 4542]. GOF: switches IIa to IIb, socket to shaft, and sheath to neuron (all = opposite of LOF) [1307, 3027, 3579, 4542, 4789]. Extreme GOF: 4 neurons [4542]. PROTEIN: Function: determinant of bristle cell fates (zygotic form: 556 a.a.) that is unequally partitioned in SOP mitoses: inherited by IIb, shaft cell, glial cell, and neuron [1447, 3579, 4417, 4542]. Location: cell cortex (i.e., inner face of cell

membrane) [1447, 3579, 4542]. Domains: PTB, PEST, zinc ﬁnger?, basic, binding sites for EH and SH3 domains [2530, 3725, 4417, 4473]. Partners: binds Partner of Numb [2609] via its PTB domain, N [1307, 1651], Nak [765, 4900], Miranda [3892, 3893], and Amos [1928]. Also binds H [4542] but not Su(H) [4542]. (Its homolog mNumb binds Seven-in-absentia [4473].)

numb-associated kinase (nak, 37B4–7). Pathway: same pathway as numb. GOF effects are sensitive to dose of numb [765]. PHENOTYPES: LOF: unknown. GOF (≈ numbLOF ): switches IIb to IIa, shaft to socket, and maybe neuron to sheath [765]. Extreme GOF (≈ numbnull ): 4 sockets [765]. PROTEIN: Function: putative serine/threonine kinase (1490 a.a.) [765]. Location: cell cortex when overexpressed [765]. Domains: putative S/T kinase domain near N terminus, and Numb-binding 11-mer peptide near C terminus [765]. Partners: binds Numb’s PTB domain [765]. prospero (pros, 86E1–2, named for the magician who sets others’ fates in Shakespeare’s The Tempest). Pathway: DS(+) of numb and DS(−) of N (and Su(H) and ttk?) in IIa: numbGOF or NLOF turns pros on in IIa daughters, while NGOF turns pros off in IIIb daughters [3550]. PHENOTYPES: Null: switches IIb to IIa (≈ numbLOF , partial expressivity); stunting of axon and dendrite [2680, 3550]. GOF: switches IIa to IIb (≈ numbGOF , ∼100% expressivity); rarely switches neuron to sheath [2680, 3550]. PROTEIN: Function: transcription factor [1753] that helps specify IIb cell identity [2680]. Prospero has two alternately spliced isoforms (1374 and 1403 a.a.) [785, 4053]. Location: IIb (but not IIa) nucleus, then basal cortex during IIb and IIIb mitoses, then nuclear in sheath cell while fading from neuron [1447, 2680, 3550]. Domains: homeo (partial), opa (2x), PEST (3x), masked NLS [1020], stretches of Ala, Ser, Thr, and Asn, plus a sequence (≤32 a.a.) that causes asymmetric localization in mitotic neuroblasts [1855, 4436]. Partners: binds Miranda [2749, 3892, 3893]. scratch (64A, scarred eye facets). Pathway: interacts with deadpan (another pan-neural gene), although its immediate US regulators are unknown [3714]. PHENOTYPES: Null: wild-type (i.e., no apparent defects) [3611]. GOF: 2 neurons (and maybe 2 sheath cells). PROTEIN: Function: pan-neural transcription factor (664 a.a.) [3611]. Location: neurons. Domains: zinc ﬁnger (5x). Serrate (Ser, 97F, serrated wing margin). Pathway: Notch pathway [4859]. PHENOTYPES: Null: wild-type in bristles. When Delta function is also eliminated, Ser null switches IIa to IIb, socket to shaft, and (less often) sheath

APPENDIX THREE. GENES THAT ALTER FATES OF BRISTLE CELLS

to neuron (≈ NLOF ) [4859]. GOF: switches shaft to socket [2008]. PROTEIN: Function: ligand for Notch receptor (1404 a.a.) [1252, 4302]. Location: transmembrane (single-pass) [1252, 4302]. Domains: signal peptide, EGF-like (14x), Cysrich, transmembrane [1252, 1943, 1944, 4302]. Partners: binds N [2263, 3544, 4601].

shibire (shi, 14A, means “paralyzed” in Japanese). Pathway: presumed to act in the Notch pathway based on bristle-pattern effects (App. 5). PHENOTYPES: Null: unknown. Null alleles exist [1595] but have not been analyzed in mosaics for effects on bristles. LOF: switches socket to shaft [1803, 3425]. GOF: unknown. PROTEIN: Function: endocytosis of Notch receptor [3271, 3863]. Homolog of mammalian dynamin. Two alternately spliced isoforms: 836 and 883 a.a. [740, 4443]. Location: cytoplasmic (free) or submembrane (clathrin-coated pits) [4549]. Domains: PH [2458, 4437], tripartite GTPase motif, and Pro/Arg-rich C terminus (ratio of ∼4 basic/acidic residues) with 5 potential binding sites for SH3 domains [740, 1095, 4396, 4549, 4802]. Partners: binds various SH3-domain proteins [1588, 3913], including Amphiphysin-2 [4655, 4656] whose 3-D structure is known [3218]. Binds itself during assembly of “collars” [954, 1852, 2184, 4240, 4549], leading to new juxtapositions of domains that may explain GTPase activation [4802]. Suppressor of Hairless (Su(H), 35B9–10). Pathway: same pathway as numb. Su(H)LOF synergistically suppresses numbLOF [4542]. DS(−) of numb: double-null ≈ Su(H)null [4542], and double-GOF ≈ Su(H)GOF [3027]. Antagonistic to H, but epistatic relationship (US vs. DS) is unclear: double mutant of H LOF and Su(H)null has mixture of H LOF and Su(H)null traits [3827]. PHENOTYPES: Null: apparently switches IIa to IIb [999, 3820], but little – if any – switching of sheath-to-neuron identity [3027]. LOF (near time of SOP mitosis): switches socket to shaft (but not sheath to neuron) [3827, 4542]. GOF: switches shaft to socket (but not neuron to sheath) [200, 3027, 3827, 4542]. Rarely switches IIb to IIa to yield a 4-socket cluster (≈ H null ) [3027]. PROTEIN: Function: transcription factor (594 a.a.) [1131, 3826]. Enhances transcription by using N as co-activator [1329, 2211] but probably blocks transcription in absence of N by recruiting a histone deacetylase complex [1913, 2129, 2941]. In adults, it acts physiologically in the socket cell to permit deﬂections of the shaft to be transduced into electrical impulses [218]. Location: nuclear in cultured cells, but

275

moves to cyt. if N is added; returns to nuc. if N binds Dl returns to cyt. if Numb is added [1307]. Also shows N-dependent movement from cyt. to nuc. in socket cells in vivo [1448]. Detectable in epidermal cells and SOP lineage (equal amounts in IIa and IIb); strong expression in socket cell after IIa mitosis [1448, 3027]. Domains: novel (2-part?) DNA-binding domain, nonfunctional integrase motif, NLS (2x) [788, 1331, 3825, 3826]. Partners: binds N [1269, 2747, 4244] and H [171, 492, 1332, 2657] but not Numb [1307, 4542]. Binds as monomer to DNA at GTGGGAA (high afﬁnity) and GTGAGAA (low afﬁnity) [788, 1332, 4408]. These sites are paired in promoters of a subset of E(spl)-C genes [171, 1131, 2453]. [1269];

tramtrack (ttk, 100D, railroad pattern of gene expression in embryonic germ band). Pathway: DS(+) of N: ttkLOF prevents NGOF from changing neuron to sheath [1651]. DS(−) of numb: double-LOF ≈ ttkLOF [1650]; ttkLOF does not alter Numb asymmetry [1650]; Ttk appears after Numb [3502]. PHENOTYPES: Null: 4 neurons (≈ extreme numbGOF ) [1650]. GOF: 2-shafts-2 sockets (switches IIb to IIa) [1650, 3502]. Excess blocks mitosis [3502]. Extreme GOF (rare): 4 sockets (≈ numb null ) [1650]. PROTEIN: Function: transcription factor, which has two alternately spliced isoforms: 69 kD (641 a.a.) and 88 kD (811 a.a.) [1650, 1731, 3537]. Location: nucleus [3502]. The 69 kD isoform is detected in IIa (not IIb), sheath (not neuron), socket and shaft cells, and slightly in epidermal cells [1650, 1651, 3502]; while 88 kD is also detected in 3 bristle cells (probably sheath, socket, shaft) in eyes but acts differently [2391, 2529]. Domains: zinc ﬁnger (C2H2 type, 2x), BTB [211, 1516, 4596], PEST [1731]. Partners: binds dCtBP (via BTB domain) [4596] and binds itself as homo-oligomer (via BTB) [211, 1516]. Isoforms bind different DNA sequences [1187, 2123, 3537]: GGTCCTGC (69 kD) or AGGGC /T GG (88 kD). twins (85F, mirror-image duplications of the wing disc). PHENOTYPES: LOF (≈ numbLOF and nakGOF ): 2-shafts-2 sockets (switches IIb to IIa) [3904]. GOF: unknown. PROTEIN: Function: regulatory subunit of serine/threonine protein phosphatase PP2A, which has two alternately spliced isoforms: 443 and 499 a.a. [2761, 4418]. PP2A dephosphorylates activated kinases in various signaling pathways [2861]. Subcellular location: unknown. Uniform in discs [2761, 4418]. Partners: binds the PP2A catalytic subunit [2861], which in turn binds Axin [1919].

APPENDIX FOUR

Genes That Can Transform One Type of Bristle Into Another or Into a Different Type of Sense Organ

In parentheses after each gene name are the salivary gland map location and the origin of the name. The PHENOTYPES section only lists bristle-related phenotypes (see Fig. 2.8). The PROTEIN section presents available data on function, length, subcellular location, domains, and binding partners. For further information, see FlyBase and The Interactive Fly. Abbreviations: CS (chemosensory), DS (downstream in causation; “+” activated or “−” inhibited by US gene), GOF (gain of function), LOF (partial loss of function), MS (mechanosensory), US (upstream). Evidence for the hierarchical order of genes in a pathway (US or DS) is given from a DS perspective only. Protein domains are deﬁned in Appendix 1. Numbers of repeats (e.g., “6x”) are in parentheses. Other genes whose embryonic mutant phenotypes suggest that they may belong to this group are BarH1 and BarH2 [1456, 1843]. Certain sensilla campaniformia on the wing are transformed to bristles by ash2 (a member of the Trithorax Group of regulators; cf. Ch. 8) [15].

absent solo-MD neurons and olfactory sensilla (amos, 36F; “MD” stands for multiple dendritic). Pathway: DS(+) of lozenge [1587]; interacts positively (dosedependent) with daughterless [1587]. PHENOTYPES: Null: unknown. LOF: fewer sensilla basiconica and trichodea on the antenna [1587]. GOF: extra sensilla basiconica, trichodea, and coeloconica on the antenna, ectopic olfactory sensilla elsewhere, and conversions of bristles into olfactory sensilla [1587]. PROTEIN: Function: transcription factor (198 a.a.) [1587, 1928]. Location: unknown (presumably nuclear). Domains: bHLH (C-terminal) [1587, 1928]. Partners: binds Daughterless [1928], and these het276

erodimers bind E boxes [1928]. Also binds Numb [1928], but the function of this interaction is unknown.

atonal (ato, 84F, missing chordotonal organs). Pathway: US(−) of cut [2038]. PHENOTYPES: Null: missing chordotonal organs (due to failure of SOP initiation) [2040, 2042]. GOF: transforms bristles into chordotonal organs [2038] and causes ectopic chordotonal organs [764, 2040]. PROTEIN: Function: transcription factor (312 a.a.) [2040]. Location: chordotonal SOPs [2040]. Domains: bHLH (C-terminal), acidic (transcriptional activation?) section [2040]. Atonal’s speciﬁcity for evoking chordotonal (vs. bristle) organs resides in its bHLH domain [764]. Partners: binds Daughterless, and these heterodimers bind E boxes [2040]. cut (7B1-2, scalloped margin of the wing). Pathway: DS(−) of ato. Expressing ato in bristle SOPs abolishes cut expression there, but ato is not activated in transformed chordotonal SOPs of cut null embryos [2038]. Was thought to be DS(+) of poxn because (1) ubiquitous poxn activates cut in SOPs where cut is not normally expressed, and (2) reporter gene driven by a piece of the cut promoter is expressed in CS (but not MS) bristles [4481]. However, other evidence (see poxn) indicates independence. PHENOTYPES: Null: partial or complete transformation of MS bristles into chordotonal organs [378]. GOF: unknown for adult bristles. In the embryo, transforms chordotonal organs into external sense organs [372]. PROTEIN: Function: transcription factor (2175 a.a.) [370], which acts as a repressor by recruiting histone deacetylases [2528]. Location: nucleus of SOP and all descendants in embryonic external sense organs [370, 371] and adult bristles (both MS and CS) [149, 2000]. Domains:

APPENDIX FOUR. GENES THAT TRANSFORM ENTIRE BRISTLE ORGANS

homeo, cut repeats (3x), highly acidic stretches of Glu and Asp (4x), regions rich in Gln (with His or Ala), Pro and Ala, Ala alone, or Asn alone [370]. Partners: binds DNA [87] and chromatin-modifying proteins [2528].

paired box-neuro (poxn, a.k.a. pox neuro, 52D1, has “paired box” DNA-binding motif and is transcribed in the CNS). Pathway: independent of cut. Surprisingly, poxn is expressed normally in SOPs of cut null embryos [4481], and cut is expressed normally in SOPs of poxnnull wings and legs [149]. PHENOTYPES: Null: transforms CS (poly-innervated) into MS (mono-innervated) bristles

277

on the dorsal tibia and wing margin [149], and suppresses some CS bristle SOPs (suggesting a proneural role). LOF: milder version of null phenotype [149], plus differentiation defects within the bristle organ. GOF: transforms MS into CS bristles on the dorsal tibia [149, 3141, 3142]. PROTEIN: Function: putative transcription factor (425 a.a.) [401,950]. Location: nuclei of CS bristle SOPs and most or all SOP descendents [149, 950, 3142]. Domains: “paired” domain, poly-Ala stretches, a Pro-rich segment (15/45 a.a.), a highly charged region (14/25 a.a.), and an acidic region (18/45 a.a.) [401, 950]. Partners: binds DNA, including the cut A3 enhancer [4481].

APPENDIX FIVE

Genes That Can Alter Bristle Number by Directly Affecting SOP Equivalence Groups or Inhibitory Fields

Some of the genes catalogued below are also listed in Appendix 3 because their mutations affect both bristle development (App. 3) and patterning (App. 5). Redundancy is minimized by stating only those PHENOTYPES here that pertain to patterns. For genes that are listed in both appendices, the reader is referred to Appendix 3 for protein domains and binding partners. See Table 6.2 for upstream “prepattern” genes that inﬂuence bristle patterning indirectly. In parentheses after each gene name are the salivary gland map location and the origin of the name, except where obvious. The PROTEIN section presents available data on function, length, subcellular location, domains, and binding partners. For further information, see FlyBase and The Interactive Fly. Abbreviations: “≈” (resembles), a.k.a. (also known as), cyt. (cytoplasm), DS (downstream in causation; “+” activated or “−” inhibited by US gene), GOF (gain of function by mutant allele or transgene construct), LOF (partial loss of function), MC (macrochaete), mC (microchaete), nuc. (nucleus), PNC (proneural cluster), SOP (sensory organ precursor), US (upstream), UTR (untranslated region). To avoid redundancy, data about hierarchical rank within a pathway are given at the DS gene only. Thus, the link “a b” would be entered under gene b as “DS(+) of a” followed by epistasis evidence, but under gene a as just “US(+) of b” without the data. Likewise, “c d” would be listed for d as “DS(−) of c” with evidence, but for c as “US(−) of d” without the data. Protein domains are described in Appendix 1. Numbers of repeats (e.g., “6x”) are in parentheses. Many of the tabulated genes perform similar duties in the embryonic CNS and PNS [2018, 2019, 2062]. Some genes

278

whose mutant alleles add or delete bristles were omitted because they act redundantly with a listed gene (e.g., mα and m4 [92]) or because their effects are too slight or too poorly studied: aristaless [3798], Dense [2500], Domina [4139], echinus [4185], l(3)ecdysoneless [3990], puckered [2713], Suppressor of deltex [1276], Tufted [102, 1802], etc. [951, 1514, 4333]. Scutoid GOF (not listed) [126] involves misexpression of Snail (a zinc-ﬁnger protein) in the scutellum, where it blocks AS-C function (i.e., suppresses SOPs) by competing for the same E boxes [1334]. The role of the pairrule gene odd Oz is uncertain: it is expressed in SOPs [2497], but no mutational effects on bristles have been reported. The only known maternal effect on bristle number is from isoalleles of tumorous head 1 [3220]. Additional pattern-affecting genes were recovered in a recent GOF screen [6]. Shaggy (Sgg) was once thought to be a downstream agent of Notch in lateral inhibition [2018, 3681, 3963], but subsequent evidence argues that it is an upstream regulator of PNCs (Table 6.2) [2890]. The old view was based on three main facts, each of which is open to other interpretations. Fact #1: Notal sgg null clones only cause extra bristles in proneural areas [1797, 3681, 3963], and this conﬁnement to PNCs suggests that sgg functions in lateral inhibition. Rebuttal: Although bristle density does increase in sgg null clones [886, 3373], the density is more similar to acGOF [1577] than to NLOF [459]. The lack of N LOF like “tufting” argues that the Notch pathway still mediates lateral inhibition despite sgg null . Moreover, sgg null clones put bristles on the wing [351, 353, 3349, 3958], so sgg must act upstream of AS-C. Fact #2: Extra doses of the AS-C do not add bristles to sgg null clones [3956], so sgg must not be affecting the proneural stage. Rebuttal: If

APPENDIX FIVE. GENES THAT ALTER BRISTLE NUMBER

the Notch pathway is still functioning in sgg null clones (as argued above), then increasing the dose of proneural agents cannot force bristles any closer than a minimum distance dictated by the inhibitory ﬁelds. The absence of an overt phenotype does not preclude a genetic interaction. Fact #3: Alleles of sgg can interact with Abruptexclass Notch (N Ax ) alleles [1797, 3681, 3963]. Rebuttal: N Ax mutations involve Fringe [990] and Wg signaling [459, 886], so this interaction could be due to Wg (and only indirectly to Notch). Wg is the likely culprit in sgg-mediated hyperplasia because the macrochaete sites most affected by sgg LOF are those that rely on wg [3373, 3374, 3956, 4368]. Conclusion: Sgg appears to affect SOP initiation solely via the Wg pathway [353, 886, 3374]. In contrast, Dsh affects SOPs by both the proneural and lateral-inhibitory routes by short-circuiting the Wg and Notch pathways [151, 459, 1054]: “Wg Dsh {Notch AS-C}.”

achaete (ac, 1B1–7, AS-C, missing bristle or “chaete” in Latin). Pathway: same proneural pathway as da [949]. DS(−) of N, Su(H), and E(spl)-C (see text): compound of ac null sc null with N LOF [997, 1797, 1802], Dl LOF [997], or E(spl)-C deletion [1794] ≈ ac null sc null . DS of H: H GOF fails to rescue ac null sc null [197]. DS(−) of h: ac is upregulated in h LOF [3982]. Paradoxically, it is also US(+) of h (see h). DS(−) of emc: ac null sc null is epistatic to emc LOF [997]. US(+) of Dl [1672, 2359] (but see Dl ), Brd, sca, E(spl)-C genes m4, m7, and m8 [2885, 3974], and klu [2250]. DS(−) of tam (a.k.a. pyd ): more Ac in tam LOF [4237]. PHENOTYPES: Null: unknown except when nearby enhancers are also deleted, in which case ac null removes many mCs and a few speciﬁc MCs [1538]. In combination with sc null , ac null eliminates nearly all bristles [1379, 1462, 1802, 3812] by suppressing SOP initiation [912]. LOF: fewer mCs and loss of certain MCs [1802, 4185]. GOF (“Hairy wing” alleles [635]): extra bristles (including mCs and MCs) on the thorax, wing blade, and legs [191, 1348, 1577, 1802]. PROTEIN: Function: proneural transcription factor (bHLH type; 201 a.a.) [4485]. Location: nucleus in PNC cells, more in SOPs [912, 3763]. Domains: bHLH, C-terminal acidic region [4485], which could activate transcription [2878]. Partners: binds certain “E box” DNA sequences as an Ac/Da heterodimer (rather than binding itself or as Ac/Sc) [588, 589, 918, 2359, 4452, 4453]. Also binds Emc but not Hairy or M8 [589, 4452]. Ac binds Pannier and Chip (as an Ac/Da dimer) in a multimeric complex that may bridge ∼30-kb gaps between E boxes and regional cis-enhancers at the AS-C [3504]. How Ac activates transcription is unknown; some other bHLH proteins do so by recruiting histone acetyltransferases [2737].

279

asense (ase, 1B1–7, AS-C, sensory defect). Pathway: DS(+) of ac and sc temporally [438]. PHENOTYPES: Null: defects in stout bristles along anterior wing margin [1079, 2039] and loss of tergital mCs [2690]. GOF: extra bristles, but these are probably artifacts of Ase mimicking Ac or Sc because l’scGOF also causes extra notal bristles despite l’sc not being transcribed in wild-type wing discs [438, 1079]. PROTEIN: Function: pan-neural transcription factor (bHLH type; 486 a.a.) [1540]. Location: nucleus in SOPs only [438, 1079]. Domains/partners: see Appendix 3. Bearded (Brd, 71A1–2, extra bristles) – one of 6 related genes in the Brd Complex, which act alike [2382, 2383, 4842]. Pathway: same pathway as N. The extra-bristle phenotype of BrdGOF is partly suppressed by an extra dose of N+ [2500]. DS(+) of AS-C: expression in wing disc PNCs is abolished in ac null sc null [3974]. US of H [2500]. PHENOTYPES: Null: wild-type (i.e., no apparent defects) [2500], probably due to redundancy [2382]. GOF: tufts at MC and mC sites (but bare areas persist between mC sites; cf. neuLOF ) or mild bristle loss, depending on allele and body region [2382, 2385, 2499, 2500]. PROTEIN: Function: unknown (81 a.a.) [2499]. Brd resembles the E(spl)-C proteins Mα, M2, M4, and M6 [92, 3075, 4767], and the 3 UTRs of their mRNAs also share motifs (“Brd,” “GY,” and “K” boxes) that suggest a common function [2499, 2500]. That function probably involves regulation of proneural genes whose mRNAs have motifs (“proneural boxes”) complementary to GY boxes in their 3 UTRs [2386]. Location: unknown; Brd is transcribed in PNCs [2499]. Domains: no obvious motifs except for one putative amphipathic α-helix [2383, 2499]. big brain (30F). Pathway: Notch pathway (see GOF data). PHENOTYPES: Null: 2–4x increase in bristle number (milder than N LOF ) [3519]. GOF: essentially wild-type, except when overexpressed concomitantly with N or Dl, with which it interacts synergistically [1075]. PROTEIN: Function: may form channels in cell membrane for exchange of small molecules (700 a.a.) [573, 3520] to aid reception of (or response to) Delta signal [1075]. Location: possibly transmembrane (6-pass) [3520], although antibodies detect it on cytoplasmic face of plasma membrane, on adherens junctions, and in vesicles [1075]. Colocalizes (intracellularly) with Dl and N in PNCs [1075]. Domains: hydrophobic stretches (6x), Gln-rich in Cterminal half [573, 3520]. Partners: binds N [4601, 4823]. canoe (cno, 82E4–F2, dorsal surface of embryo remains open). Pathway: Notch pathway based on synergistic interaction with N LOF , but probably also used in Ras signaling because Cno binds Ras [2363]. Also, cnoLOF

280

interacts with sca [2882]. PHENOTYPES: LOF: extra MCs [2882]. GOF: unknown. PROTEIN: Function: intercellular signaling (1893 a.a.) [652, 2882]? Location: intercellular (adherens) junctions [4236]. Domains: PDZ, Ras-binding, kinesin-like, myosin (class V)-like [2882, 3417, 3418, 4236]. Partners: binds Pyd [4236] and Ras [2363].

C-terminal Binding Protein (CtBP; 87D7–9). Pathway: dose-sensitive interactions with h [3430]. PHENOTYPES: LOF: extra bristles (MCs) on the scutellum, other parts of the notum, and head [3430]. GOF: unknown. PROTEIN: Function: regulates Hairy’s repression of AS-C (386 a.a.) [3430], probably as a co-repressor like Groucho (cf. Fig. 3.12e) [2129, 3112, 4213]. However, dCtBP can also behave as a co-activator in other contexts [3377]. Location: not known for discs. Domains: (unmapped) dimerization and peptide-binding domains [3430]. Partners: binds itself, PLSLV in Hairy, and PVNLA in E(spl)-C’s Mδ (but not M8, Emc, or Gro) [3430]; binds ¨ PxDLS in Knirps, Kruppel, and Snail [3112, 3113]. Cyclin A (CycA, 68E). PHENOTYPES: LOF (due to Pelement insertion in ﬁrst intron): removal of speciﬁc MCs (DC, SC, etc.), socketless shafts, shaftless sockets, and other defects indicative of an insufﬁcient number of mitoses [4415]. GOF: induces cells to enter S phase [4047], but effects on bristle patterning (aside from ommatidial defects [1087]) have not been reported. PROTEIN: Function: regulates G2 /M transition [2270]. Degradation of Cyclin A at metaphase allows cells to enter mitosis [2482, 4415, 4633] (491 a.a.) [2481]. Location: PNCs, less in background epidermal cells [2225]. In embryos, CycA associates with spindle poles and chromatin during mitosis (up to metaphase) but is cytoplasmic in interphase [2481, 2664]. Domains: Cdc2-binding “cyclin-box” domain, “destruction box” motif (needed for ubiquitination and degradation) [975, 1512, 3009, 3926]. Partners: binds Roughex [143], Cdc2, and other cyclin-dependent kinases [1793]. daughterless (da, 31E). Pathway: same proneural pathway as ac and sc [949]. DS(−) of E(spl)-C: m8 and m5 reduce Da activation of reporter gene [3171]. US(+) of Dl [2359]. PHENOTYPES: Null: unknown for discs. In embryos danull blocks all PNS development [688] after SOP emergence [4435] by preventing mitosis [1755]. LOF: in presence of heterozygous AS-C deletion, haploid dose of da+ removes certain MCs [949]. GOF: unknown. PROTEIN: Function: proneural transcription factor (bHLH type; 710 a.a.) [689, 906]. Da is a ubiquitous partner for AS-C proteins (cf. Tango [4022]). Location: nucleus; ubiquitous

APPENDIX FIVE. GENES THAT ALTER BRISTLE NUMBER

in discs [905, 4435]. Domains: bHLH, leucine zipper, PRD (2x), opa (2x), poly-G (2x), poly-A, PEST [689, 906]. Partners: binds certain “E box” DNA motifs as a heterodimer with bHLH AS-C proteins (Ac, Sc, L’Sc, Ase) or, less so, as a homodimer [588, 589, 918, 2039, 2359, 3171, 4452, 4453]. Also binds Emc [589, 4452], Amos [1928], and – under some [68] but not other [4452] conditions – various E(spl)-C proteins. Binds Chip (as a Da/Ac or Da/Sc dimer) in a complex with Pannier (notum only) [3504]. May also bind Net [2741].

Delta (Dl, 92A2, delta-shaped tips of wing veins). Pathway: US(+) of N [2542], but DlLOF suppresses Abruptex alleles (see text) [992, 1798, 4780], and Dl is downregulated by NGOF [3270]. Equivocal with respect to AS-C: DS(+) of AS-C (ectopic expression of sc activates Dl transcription in congruent areas of wing disc [1854]) but also US(−) of AS-C (DlLOF upregulates ac [3270], and acnull sc null is epistatic to Dl LOF [997]). Interacts with sca (see sca). PHENOTYPES: Null (assayed in mosaics): extra bristles (tufts at MC sites and high-density mCs) [2690, 4859]. LOF: t.s. LOF alleles cause extra bristles when mutants are heat-pulsed at a certain time but bristle loss (due to fate switches) when pulsed later [1797, 3272, 3273, 3277]. Haplo-insufﬁciency causes mild increase in bristle density [997, 4466]. GOF: unknown. PROTEIN: Function: ligand for Notch receptor (832 a.a.) [3022]. Serrate is not a redundant ligand in PNCs [4859]. See Figures 2.2, 2.3, and 3.6. Location: transmembrane (single-pass) [2300, 4465] but also secreted under some conditions [2264, 3479]. Detected on surfaces and in cytoplasmic vesicles of cells in PNCs [2296] and notal stripes [3270]. Domains/partners: see Appendix 3. deltex (dx, 6A, Delta-like gene on the X). Pathway: US(+) of N [2746]. PHENOTYPES: Null: unknown [580, 1053]. LOF: extra bristles and disrupted patterns [1570]. GOF: no effects on bristle number reported. PROTEIN: Function: unknown (737 a.a.) [580]. Location: cytoplasm [580, 1053]. Domains/partners: see Appendix 3. Dichaete (D, 70D1–2, a.k.a. ﬁsh-hook, missing MCs). Pathway: interacts with AS-C: D GOF is partly suppressed by certain scLOF alleles [2561]. PHENOTYPES: effects of D inversions on bristles are probably not actually due to D but rather to breakpoints in mirror (Iro-C; cf. Table 6.2) [3079]. LOF: unknown (with regard to bristles) because mutant cells become necrotic [2977]. GOF: removes certain MCs [3405, 3707, 4178]. PROTEIN: Function: activates transcription directly [2635] and may sterically facilitate DNA binding by other transcription factors (382 a.a.) [3045, 3707]. Location: nucleus [2635]. Domains: Sox (a subgroup of HMG), NLS (within Sox), short

APPENDIX FIVE. GENES THAT ALTER BRISTLE NUMBER

stretches of Ala, Gln, or Ser, plus 11 repeats of a 5-a.a. consensus [2977, 3045, 3707]. Partners: binds AACAAT and AACAAAG [2635].

Enhancer of split (E(spl), a.k.a. m8, 96F11–14, enhances split, a N LOF allele) – one of 7 bHLH genes in the E(spl)-C that act alike in many contexts [3075] (but see [871, 2548, 2549]). Pathway: DS(+) of N: M8 levels are lower in N LOF and higher in N GOF [2052]. DS(+) of kuz: m8 GOF rescues kuz LOF [4025]. DS(+) of Su(H): Su(H)-binding sites in m8 promoter are needed for expression of m8 in PNCs (but not in SOPs) [2453]. US(−) of da [3171], ac, and sc [918, 991, 1794, 2063]. Paradoxically, it is also DS(+) of AS-C: expression in PNCs is abolished in ac null sc null [3974] and augmented in scGOF wing discs [871]. Also regulated by other pathways [3075, 4568]. PHENOTYPES: Null: wild-type, due to redundancy of other E(spl)-C genes [1018, 1794, 3806]. Deletion of all 7 bHLH E(spl)-C genes causes extra bristles (tufts at MC sites and high-density mCs) [999, 1794]. GOF: missing bristles [2384, 2548, 3037], due to stiﬂing of SOP initiation [3037, 4256]. PROTEIN: Function: transcription factor (bHLH type; 179 a.a.) [2246, 2274, 3171]. Location: nucleus of non-SOP PNC cells [871, 2052, 2053]. Domains/partners: see Appendix 3. Epidermal growth factor receptor homolog (Egfr, 57F, a.k.a. DER, torpedo). Pathway: EGFR pathway, but also interacts with Notch pathway [3465, 3634]. PHENOTYPES: LOF: loss of MCs from ocellar, supra-alar, humeral, and posterior postalar sites (the scutellum is unaffected) but extra bristles at postvertical and anterior postalar sites [814]. Whether this spatial heterogeneity stems from Egfr expression or ligand access is unknown. Also causes slight (∼20%) increases in mC density [1042]. GOF: unknown. PROTEIN: Function: receptor tyrosine kinase (1459 a.a. vs. 1410 a.a. for the Type-1 vs. -2 splicing variants) [813]. Location: transmembrane (single-pass) [813]. During the larval period, Egfr is transcribed in imaginal discs [2125, 3779, 3928] and in nondividing (polytene) larval tissues [2152]. Egfr is expressed in eye discs [4843]. Domains: signal peptide, Cys-rich extracellular (ligand-binding) region, transmembrane, tyrosine kinase [813, 2573]. extramacrochaetae (emc, 61D1–2). Pathway: interacts with AS-C (see text and Fig. 3.12) and with genes of the Notch pathway [997, 1349, 2690, 3982, 4453]. DS(+) of tam (a.k.a. pyd): less emc RNA in tamLOF , but tam RNA is normal in emc LOF [4237]; synergistic effect on bristles in emc LOF tam LOF double mutant. PHENOTYPES: Null: extreme LOF defects [1349, 2690]. LOF: extra bristles (mainly MCs) at normal and ectopic sites on thorax and wings [997, 1349, 2959] and conversion of mCs to MCs on ter-

281

gites [2690]. GOF: absence of speciﬁc MCs and reduced number of mCs [997, 1349, 2690, 4453]. PROTEIN: Function: antagonist of proneural genes (199 a.a.) [1156, 1388]. Also involved in cell proliferation [206]. Location: expressed unevenly throughout the wing disc (cf. Fig. 6.2) [913, 1156, 4453]. Domains: HLH (but no adjoining basic domain), Glnrich C-terminal region [1156, 1388]. Partners: binds Ac, Sc, and Da [68, 3430, 4453].

fasciclin II ( fasII, 4B1–2, glycoprotein on certain axon fascicles in embryonic CNS). Pathway: fasII LOF phenotypes are enhanced by LOF mutations in the Ableson tyrosine kinase gene (even when the latter are heterozygous) [1351], although the latter mutations cause no such phenotypes on their own. PHENOTYPES: Null (assayed in mosaics): missing MCs on the head (verticals, postverticals, ocellars, but not orbitals) and notum (and reduced density of mCs) due to failure of AS-C activation needed for SOP initiation [1351]. Also missing ocelli (due to absence of atonal expression). LOF: similar to null phenotype but less extreme. GOF: extra bristles on the head (near the postverticals) and other regions (including scutellum) [1351]. PROTEIN: Function: neural cell adhesion molecule (two splicing isoforms: 811 vs. 873 a.a.) [1620, 2553, 4757, 4829]. Location: expressed throughout the eye disc, most intensely in the morphogenetic furrow and in the prospective ocellar region [1351]. Domains: signal peptide, Ig-like (C2-type; 5x), ﬁbronectin (type III [1956]; 2x), transmembrane (873-a.a. isoform only) [1620, 1725]. Partners: Binds itself to mediate homophilic adhesion [1619, 1620]. groucho (gro, 96F11–14, extra bristles like bushy eyebrows of comedian Groucho Marx). Pathway: DS of H: tufting trait of gronull is epistatic to balding trait of H null in double-mutant clones [197]. Interacts synergistically with h [1794]. PHENOTYPES: Null: extra bristles (tufts at MC sites and high-density mCs) with virtually no intervening epidermal cells [997, 1794, 2690]; ectopic bristles on scutellum and wing (due to interaction with h) [1794]. LOF: extra bristles near the eyes [3265]. GOF: unknown. PROTEIN: Function: co-repressor (719 a.a.) [1746]. That is, it inhibits transcription via a DNA-binding protein that tethers it to DNA [1243]. Can silence transcription at long (≥1 kb) range [4865], apparently by multimerizing [738] and recruiting histone H3 [3232]. Location: nuclear [692, 4255]. Domains: WD (7x), leucine-zipper-like sequences (2x in Pro-rich N-terminal region) that mediate the tetramerization needed for Gro to repress transcription [738, 1243, 1746, 3392]. Partners: binds itself [738], Drosophila Histone H1, and histone deacetylase Rpd3 to form a

282

complex that apparently self-polymerizes and spreads to silence chromatin [737]. Gro is recruited to DNAbinding proteins by various motifs, including (1) WRPW at C-termini of Hairy, Deadpan, and E(spl)-C bHLH proteins [3278, 3430]; (2) WRPY at C-termini of proteins that have a Runt Homology domain [107, 1243, 4616]; (3) FRPW near the N-terminus of Huckebein [1526]; and (4) LFTIDSILG in dGoosecoid [2066] and a related motif in Engrailed (cf. Fig. 3.12e) [1243, 2064, 4351]. Gro also binds Dorsal [1110, 1243] and dTcf [692, 2496].

hairy (h, 66D15, extra bristles). Pathway: US(−) of ac (see ac) but also DS(+) of ac and/or sc: downregulated in double-null for ac and sc [3982] (see text and Fig. 3.12). PHENOTYPES: Null: extra mCs and misalignments [3193]. LOF: extra mCs in various regions [1802, 1981, 2561, 2959]. GOF: ubiquitous expression of hs-hairy+ transgene erases ectopic bristles caused by h LOF or ac GOF , but (surprisingly) does not alter the normal pattern [3697]. Paradoxically, hs-hairy+ induces extra bristles in the eye [507]. PROTEIN: Function: transcription factor antagonist for proneural genes (bHLH type; 337 a.a.) [3697]. Location: nucleus [663, 3697]. Domains: bHLH, WRPW (Grouchobinding) motif, “Orange” (Sc-repressing?) domain, NLS (3x), opa, Ala stretch, Pro- and Ser-rich carboxy half [976, 3430, 3697]. Partners: binds “N box” (but not “E box”) DNA sequences [4452], Gro [1242, 3430], and CtBP [3430] but not Ac or Sc [589, 3430, 4452]. However, its ability to bind CtBP may be nonfunctional [4865]. Hairless (H, 92E12–14, bristle loss). Pathway: antagonistic to Su(H), but epistatic relationship is unclear: double mutant of H LOF and Su(H)null has mixture of H LOF and Su(H)null traits [3827]. Similar antagonism (but equivocal epistasis) to N and E(spl)-C [171, 197, 437, 2627]. DS of Brd: compound of H LOF and Brd GOF looks like H LOF [2500]. US of gro, ac, and sc [197]. PHENOTYPES: Null (≈ Su(H)GOF ): balding due to failure of SOP initiation [198]. GOF (≈ Su(H)LOF ): extra bristles (milder than N LOF ) [2657] or balding (SOP status unknown), depending on stage when transgene is overexpressed [200]. PROTEIN: Function: antagonist for Su(H). Thought to act by blocking Su(H)’s DNA-binding domain (1059 a.a.) [200, 492, 2659], H also recruits the co-repressor dCtBP [1329] ([2129] sequel). Location: nucleus [2657, 2658]; H mRNA is ubiquitous in discs [200] as is H protein [2658]. Domains/partners: see Appendix 3. klumpfuss (klu, 68A1–2, means “clubfoot” in German, refers to leg defect). Pathway: DS(+) of AS-C: kluLOF interacts synergistically with ac LOF for bristle loss and is epistatic to acGOF . PHENOTYPES: LOF: partial loss of MCs

APPENDIX FIVE. GENES THAT ALTER BRISTLE NUMBER

(head and notum) and other bristles (wing margin and legs). PROTEIN: Function: putative transcription factor (750 a.a.) [2250]. Location: PNCs, but only transiently expressed in SOPs. Domains: zinc ﬁnger (C2H2 type, 4x), putative NLS (3x), stretches of poly-A (3x) and poly-N (3x), stretches rich in Q, H, or P near N-terminus, and tandem collagen-like repeats (4x) of G(R or K)E.

kuzbanian (kuz, 34C4–5, named for a popular puppet with tufts of hair). Pathway: US(+) of N [3239, 4025] and US(+) of m8 [4025]. PHENOTYPES: Null (assayed in mosaics): absence of bristles within a clone but dense tufts of bristles when a clone border skirts a MC or mC site [3640]. LOF: extra bristles (tufts at MC sites and highdensity mCs) but also patchy bristle loss [4025]. GOF: unknown. PROTEIN: Function: protease (1239 a.a.) for Delta [3479, 3640] and probably also for Notch [2298, 3153, 3239] (see [3479] sequel for debate). Location: transmembrane (single-pass) [3640]. Expressed ubiquitously in imaginal discs [4025]. Domains: ADAM, signal peptide, prodomain, Cys-rich, transmembrane [3640]. mastermind (mam, 50C20–23, enlarged CNS). Pathway: Notch pathway [1000, 1514, 2394, 4780], possibly US of Delta [1816]. PHENOTYPES: LOF (as deduced from constructs that act in a dominant-negative manner): extra MCs (due to more SOPs) on the notum (≈ N LOF ) [1816]. GOF: unknown. PROTEIN: Function: possibly a transcription factor (1596 a.a.) [1816, 4007] or co-activator for Notch [3355, 4812]. Location: nuclear; ubiquitous in leg and wing discs [305, 3787, 4007]. Domains/partners: see Appendix 3. neuralized (neu, cf. its enhancer trap “A101,” 85C, neural hyperplasia in the embryo). Pathway: Notch pathway, but not for wing veins or margin [2387]. US(+) of N [1000, 2542]. PHENOTYPES: Null: balding [1058] due to 4neuron phenotype [2387, 4814]. LOF: extra bristles (tufts at MC and mC sites ≈ Brd GOF ) [2387, 4814]. Initially, it was thought that the extra SOPs arise autonomously via a failure of lateral inhibition [2387, 4814]. However, recent evidence demonstrates nonautonomy ([2387] sequels). GOF: balding due to suppression of SOPs [2387]. Extreme GOF: extra bristles (tufts at MC and mC sites) apparently due to a dominant-negative effect of Neu at high doses [2387]. PROTEIN: Function: ubiquitin ligase (754 a.a.; see [2387] sequels) [417, 3463]. Expressed in SOPs, but neither neu transcripts [2387] nor the A101 enhancer trap are detectable in other PNC cells [417, 1925, 2387], so Neu may only be needed in SOPs to inhibit other PNC cells. Location: cell membrane [2387, 4814]. Domains: see Appendix 3.

APPENDIX FIVE. GENES THAT ALTER BRISTLE NUMBER

Notch (N, 3C7, notched wing tip). Pathway: DS(+) of Dl: double mutant of NGOF and DlLOF ≈ NGOF [2542] (but see Dl ). DS(+) of dx: N GOF rescues dx LOF phenotype [2746]. DS(+) of kuz and Psn: expressing N GOF in kuz LOF or Psn LOF background gives N GOF phenotype [4025, 4810]. DS(+) of neu: N GOF rescues neuLOF [1000, 2542]. US(+) of E(spl) [2052, 2542]. US(−) of AS-C [918, 1797, 1802, 3270, 3983]. Interacts with sca (see sca). PHENOTYPES: Null (assayed in mosaics): balding [996, 1058, 1797, 2690] (presumably due to 4neuron phenotype [1742]), plus extra SOPs [3689] (presumably due to lack of SOP inhibition). LOF: extra bristles (by blocking SOP inhibition) in heterozygotes, although one LOF allele removes bristles (by stiﬂing PNCs?) [460]. Heat-sensitive LOF alleles cause extra bristles when mutants are heat-pulsed at a certain time but bristle loss (due to fate switches) when pulsed later [603, 1742, 1802, 3689, 3891]. Haplo-insufﬁciency causes mild increase in bristle density [460, 997]. GOF: fewer bristles [2690] or balding [996, 997] due to failure of SOP initiation [197, 460, 3863]. PROTEIN: Function: receptor for Delta and signal transducer (2703 a.a.) [2210, 3022, 4611]. (Serrate does not participate in lateral inhibition [4859].) See Figures 2.2, 2.3, and 3.6. Location: transmembrane (single-pass) [2210, 4611]; ubiquitous in discs [2070, 2296]. Domains/partners: see Appendix 3. Notchless (Nle, 21C7–8). PHENOTYPES: Null: phenotypically wild-type, although Nle null /+ enhances bristleloss phenotype of a N GOF (Abruptex) allele [3662]. GOF: slight reduction in bristle number. PROTEIN: Function: possible adaptor for multiprotein complex that includes Notch [3662]. Location: unknown. Domains: WD (9x) [3662]. Partners: binds Notch [3662]. polychaetoid (pyd, 85B6, extra MCs, a.k.a. tam [4236, 4237]). Pathway: US(+) of emc; US(−) of ac [4237]. LOF alleles interact synergistically with acGOF and hLOF [1802, 3068, 3069], as well as (in dose-dependent manner) with LOF alleles of N, Dl, and emc [733]. Because pyd LOF mainly affects PNCs, pyd likely acts in or with the Notch pathway. PHENOTYPES: Null: extra MCs and mCs (due to more SOPs) on the notum [733]. LOF: extra bristles on notum [3066, 3067, 4237] and legs [1802]. GOF: unknown. PROTEIN: Function: probably intercellular signaling (two isoforms: major = 1367 a.a.; minor = 1289 a.a.) [533, 1061, 4237, 4574], possibly via Ras modulation [4792] of gap junctions [1476] or direct action in the nucleus [1181, 1576, 1920]. Location: intercellular (adherens and septate) junctions [2148, 4236, 4237] and possibly the nucleus [1576]. Localization to junction vs. membrane is controlled by RNA splicing [4574]. Domains: PDZ (3x), SH3, GUK, and

283

a Pro-rich C-terminal region. Homologous to mammalian ZO-1 [4237] and member of the MAGUK family of scaffolding proteins [1062]. Partners: binds Canoe [4236] and dCortactin (which binds actin ﬁlaments) [2148]. Its homolog (ZO-1) binds a gap junction protein (Connexin43) [1476].

Presenilin (Psn, 77A–C, refers to senility in humans afﬂicted with Alzheimer’s disease). Pathway: US(+) of N [712, 4810], although it can also affect Wingless signaling [891, 3124]. PHENOTYPES: Null: expanded PNCs where ∼all PNC cells become SOPs, apparently due to failure of lateral inhibition [4810], similar to Su(H)null . GOF: unknown. PROTEIN: Function: needed for ligand-dependent proteolytic processing of Notch [4156, 4810] and for its subcellular localization [1652], although whether Psn acts enzymatically or indirectly is unclear [1723, 2602, 3533, 4162, 4710] (541 a.a.). Location: vertebrate homolog localizes to adherens junctions [1432], as well as to endoplasmic reticulum and Golgi membranes [4710]. Domains: transmembrane (8x) [4710]. Partners: binds Notch [3533, 3534] and β-catenin (Armadillo ortholog) [86, 3003, 4060]. scabrous (sca, 49C2–D4, rough eyes). Pathway: interacts with N and Dl [179, 437, 1921, 2885, 3490] at the level of the receptor [2462] but may not be in the Notch pathway per se [186, 2461]. DS(+) of AS-C: expression in wing disc PNCs is abolished in ac null sc null [2885, 3974] (see text). PHENOTYPES: Null (same as LOF): extra bristles (usually doublets at normal sites) [1921, 2885]. GOF: unknown for bristles, but overexpression in the eye disrupts spacing of R8 cells, which are patterned like bristle SOPs [1158, 2461]. PROTEIN: Function: probably a diffusible SOPinhibitor (774 a.a.) [1921, 2885], but may act redundantly [1158, 2885]. Sca receptor is unknown [2461]. Location: extracellular (secreted) [2461] and in intracellular vesicles [186, 1921]. Domains: signal peptide, ∼200-a.a. “FReD” (Fibrinogen-Related) domain (seen in many secreted proteins) near C-terminus [1921, 2463] linked to dimerization domain by Pro-rich region. Sca is secreted as a soluble glycoprotein dimer (like many growth factors) held together by disulﬁde bonds [2461]. Partners: binds heparin [2461]. Does not bind N [2460, 4601, 4823] but associates with it in a complex [3456], so a third protein must serve as a bridge to attach them. scute (sc, 1B1–7, AS-C, loss of MCs from scutum – i.e., dorsal thorax). Pathway: same proneural pathway as da [949]. DS(−) of emc: sc is upregulated in emcLOF [912, 3982] (see emc). DS(−) of H, N, and E(spl)-C (see ac). US(+) of Dl [1672, 1854] (but see Dl ), Brd, sca, and E(spl)-C genes

284

m4, m7, and m8 [2885, 3974]. PROTEIN: Function: proneural transcription factor (bHLH type; 345 a.a.) [4485]. Location: nucleus in PNC cells, more in SOPs [912]. PHENOTYPES: Null: removes certain MCs [1538]. Compound with ac null removes nearly all bristles (see ac). LOF: removes allele-speciﬁc subsets of MCs [1453, 1538] and chemosensory bristles on legs [1802]. GOF: extra MCs [912, 918, 2038]. Domains: bHLH, C-terminal acidic region [4485], which could activate transcription [2878]. Partners: binds certain “E box” DNA sequences as a Sc/Da heterodimer (rather than binding itself or as Ac/Sc) [588, 589, 918, 2039, 4452, 4453]. Also binds Emc and several E(spl)C bHLH proteins but not M8 or Hairy [589, 3430, 4452]. Sc binds Pannier and Chip (as a Sc/Da dimer) in a multimeric complex that may bridge ∼30-kb gaps between E boxes and regional cis-enhancers at the AS-C [3504].

senseless (sens, 85D, loss of sensory neurons). Pathway: same proneural pathway as da and the AS-C [3127], although its US vs. DS rank is ambiguous. PHENOTYPES: a.k.a. Lyra, whose elimination of wing margin [2, 4] is attributable to a GOF (ectopic) activation of sens [3127]. LOF: balding [3127] due to interference with SOP survival or differentiation (or both). GOF: extra bristles within and outside regions that normally contain bristles [3127]. PROTEIN: Function: activator of proneural genes and mediator of cross-talk with the Notch pathway (541 a.a.) [3127]. Location: nuclear in SOPs of the PNS but wanes in SOP descendants [3127]. Sens is manifest after proneural gene products accumulate but before the pan-neural marker A101 (neu) is detectable. Domains: zinc ﬁn-

APPENDIX FIVE. GENES THAT ALTER BRISTLE NUMBER

ger (C2H2 type, 4x), NLS [3127]. Partners: binds the nucleotide sequence TAAATCAC, which is found upstream of three of Sens’s supposed target genes (ac, sc, and m8) [3127].

shibire (shi, 14A, means “paralyzed” in Japanese). Pathway: Notch pathway: phenotypes and sensitive periods of shi LOF ≈ N LOF [1803, 3425, 3863, 3891], and shi LOF N LOF double mutant is more affected than single mutants [3863], but unclear whether shi is US or DS of N [3863]. PHENOTYPES: LOF: extra bristles (tufts at MC sites and high-density mCs) [1802, 1803, 3425]. GOF: unknown [2561]. PROTEIN: Function: endocytosis of Notch receptor [3271, 3863], but it is unclear is why endocytosis is needed for Notch signaling [2320]. Two alternately spliced isoforms: 836 and 883 a.a. [740, 4443]. Location: cytoplasmic (free) or submembrane (clathrin-coated pits) [4549]. Domains/partners: see Appendix 3. Suppressor of Hairless (Su(H), 35B9–10). Pathway: Notch pathway. Chimeric construct of Su(H) plus VP16 activation domain mimics N’s effects [871]. Antagonistic to H, but epistatic ranking (US vs. DS) is unclear: double mutant of H LOF and Su(H)null has mixture of H LOF and Su(H)null traits [3827]. PHENOTYPES: Null: extra MCs [3820] due to extra SOPs per PNC [3826]. GOF: balding due to failure of SOP initiation [3827]. PROTEIN: Function: transcription factor (594 a.a.) [1131, 3826]: coactivator with N [1329, 2211] but repressor sans N [1330, 2129, 2941]. Location: nucleus in PNCs, less in background epidermal cells [1448]. Equal amounts in SOP vs. non-SOP PNC cells [1448]. Domains/partners: see Appendix 3.

APPENDIX SIX

Signal Transduction Pathways: Hedgehog, Decapentaplegic, and Wingless

Three of the 5 chief transduction pathways used by discs are outlined below and diagramed in Figure 5.6. The other two cardinal pathways – Notch and EGFR – are discussed in Chapters 2 (Fig. 2.2) and 6 (Fig. 6.12), respectively. Abbreviations: “MF” (morphogenetic furrow), “vh” (vertebrate homolog).

1. Hedgehog signaling pathway [1554, 1974, 2075, 2791, 3685] (see [2832] for overview). Although the agents below constitute the standard version of this pathway, notable deviations have been found [1554, 3077, 3176, 3739, 4282]. However, the heretical proposal that Hh controls target genes in Bolwig’s organ without employing Cubitus interruptus [4216] has been refuted [2834]. Aside from the components below, the zinc-ﬁnger protein Combgap regulates the levels of Cubitus interruptus in leg, wing, and eye discs [621, 4217] but acts in the Wingless pathway in optic lobes [4017]. Another possible player is oroshigane (unknown role), which acts upstream of patched [1171]. Other genes have been implicated [937, 1683, 2086] (e.g., shifted [2858]), but how their products ﬁt into the chain remains to be determined. 1.A. Hedgehog (Hh, named for the lawn of spiky denticles in LOF embryos [1974, 2893, 3151]) is a 471-a.a. polypeptide (nascent form) [2467, 2895, 4254] that cleaves itself (between residues 257 and 258) into C- and N-terminal moieties [2466, 3338, 3433, 4246]: “HhC ” bears the catalytic active site [2466], whereas “HhN ” is the signaling fragment [1231, 3433]. HhC is soluble. So is HhN , but to a lesser extent because the cholesterol tail that it acquires during the autoproteolysis reaction [255, 2705, 3432, 3434] confers an afﬁnity for membranes, Patched, or both [255, 572, 1973, 4275]. HhN also has to be acylated on an N-terminal cysteine

to be active [2464]. Diffusion of HhN is impeded by extracellular heparin [2466], and HhN requires a speciﬁc subset of proteoglycans (processed via Tout-velu and Sulfateless [277, 4275, 4375]) in order to traverse cells. Without its cholesterol tail, HhN escapes this need and travels 5 times farther but is less able to turn on target genes [572, 4275]. When Hh is artiﬁcially tethered to cell membranes, it stills convey signals but only to adjacent cells [572, 4136]. Unlike vertebrates, the ﬂy has no hh paralogs [3674]. In thoracic discs, hh is expressed exclusively in the P compartment [2467, 2832, 4227, 4228, 4254].

1.B. Patched (Ptc, name based on denticle pattern) is a 1286-a.a. transmembrane protein [644, 1895, 3036] that serves as the canonical Hh receptor [754, 755, 2691], although other Hh receptors may exist [3506]. Hh’s cholesterol tail is evidently bound by 5 of Ptc’s 12 transmembrane spans that comprise its sterol-sensing domain (SSD) [255, 572]. This domain also seen in Dispatched, a protein that interacts with Hh’s tail to enable Hh to detach from the cells that make it [572]. Ptc’s binding and signaling functions are likely mediated by separate domains [255] because mutations can block signaling without affecting binding [754], and this effect can also be achieved by C-terminal truncation [2073]. This uncoupling is shown by mutations in Ptc’s SSD that derepress Smo without altering Ptc’s ability to sequester Hh [4174]. Such mutations interfere with the “Ptc Smo” link (see below) by altering the endocytic trafﬁcking of Ptc [2711]. Ptc is an unusual receptor insofar as (1) it actively represses the pathway in the absence of ligand [1982, 3487] and (2) it negatively controls its own expression [1828, 1973, 2072, 4136]. Transcription of ptc is conﬁned to the A compartment 285

286

[3372]. In P cells, en

APPENDIX SIX. SIGNAL TRANSDUCTION PATHWAYS

ci so that Ci cannot activate ptc [65, Ptc is upregulated in border cells that receive Hh [642, 754, 1841, 1982, 2074]. The purpose of this upregulation was thought to be the boosting of responses to Hh, and responses are indeed sensitive to relative levels of Hh vs. Ptc [2072]. However, the “Hh Ptc” link serves mainly to sequester Hh and prevent its long-range diffusion [277, 572, 754, 755, 2074]. In the absence of Hh, Ptc inhibits Smo in a stable receptor complex [4130] (but see [2073]), although Ptc has a high turnover rate [2072]. Whether the “Ptc Smo” link is direct or indirect remains unclear [1022]. When Hh binds Ptc, the Ptc-Smo complex changes shape [1973, 1980] so that Smo is no longer repressed [755, 2074]. This derepression is correlated with (1) an increase in Smo phosphorylation, and (2) a dissociation of the Ptc-Smo complex, whereby Ptc and Hh enter the endocytic pathway (for lysosomal destruction?) and Smo stays at the surface [1022, 3506] (but see [4881]).

restrain Ci from entering the nucleus by tethering it to the cytoskeleton [3612, 3685, 3976] in the same way that Numb is supposed to tether Notch to the membrane (cf. Fig. 2.2). Consistent with this “Handcuff Scenario,” Cos2 interacts with Ci in a saturable, stoichiometric way [4540], and cytoplasmic retention is abolished by deleting the C-terminal domain of Ci that binds Cos2 (a.a. #941– 1065) [4536]. In wing discs, cos2 mRNA is uniform, but Cos2 accumulates (in a Hh-independent way) in the A compartment [3976]. Cos2 is phosphorylated when cells receive a Hh signal, but whether this event is causally related to the release of the complex from microtubules is not known [3612]. Also unknown is the kinase [2075], which (based on mutational dissection) cannot be Fused – thus precluding one obvious scenario (viz., Fused phosphorylates Cos2 so that it cannot bind microtubules [1974, 4536]). Releasing a transducer from microtubules may also be a step in the Dpp pathway [1084].

1.C. Smoothened (Smo, named after embryo defect) is a 1028-a.a. transmembrane (7-span) protein [61, 4441] that functions downstream of Ptc [61, 754, 1264, 1894, 2993, 3487]. Because of its resemblance to G protein-coupled receptors, Smo was initially thought to be the Hh receptor [61, 4441], but subsequent work showed that Hh physically interacts with Ptc instead [754, 755]. The evidence implies that Ptc and Smo interact as receptor (Ptc) and coreceptor (Smo), although Smo can activate the pathway by itself [1022, 1974]. Ptc appears to regulate Smo catalytically (vs. stoichiometrically) [1980, 2117], but how Smo relays the signal is unclear [2116, 2117]. Smo may be a Frizzledtype receptor that has evolved the ability to activate itself without a ligand [755]. No bona ﬁde G proteins have yet been implicated [2074] (but see [1893]). Smo is expressed in both A and P compartments of wing discs [2832] along basolateral cell surfaces [1022]. Smo appears to be degraded by a PKA-dependent route that is blocked in the presence of Hh [62], and in this way Hh evidently ampliﬁes the perceived volume of its signal [2117].

1.E. Fused (Fu, inaccurately named for fused wing veins [1198]) is an 805-a.a. protein with the sequence hallmarks of a serine-threonine kinase [365, 3460, 3920, 4280, 4281]. Its catalytic domain is needed for signal relay [3461, 4280], but its substrates are unknown [2906, 4536]. Fu homodimerizes [116] and afﬁliates with the Cos2-Ci complex [4068]. Like Cos2, it is phosphorylated [3506, 4283] when the complex detaches from microtubules in response to Hh [3612, 4283]. Fu and Cos2 can enter the nucleus, but their entry is not regulated by Hh [2833]. Transcription of fu appears uniform in leg and wing discs [4281], but Fu is more intense in the A compartment [78]. Genetically, fu acts upstream of ci as a positive regulator [78, 1264, 2967, 3341, 3991], but the effect of Fu on Ci may occur by a separate route from either Cos2 [1264, 3461, 3739] or PKA [1554, 1701, 1974, 2074, 3739]. Paradoxically, fuLOF inhibits Hh signaling while increasing the amount of detectable Ci-155, although reports differ as to whether the excess Ci-155 arises at the expense of Ci-75 [3177, 4539]. Fu might be converting Ci-155 to a form that is highly active but labile and hence less detectable [3177, 4536, 4809], although the increase could be a trivial side effect of less Fu lower response to Hh less Ptc in border zone greater diffusion range of Hh more cells expressing Ci-155 [4539]. Alternatively, Fu may just be the key that unlocks the handcuffs in the Handcuff Scenario [2479, 2833]. The Fu-Ci stoichiometry is consistent with the latter idea [4540], as is the otherwise odd fact that fu’s LOF and GOF phenotypes are similar [116].

1647, 1828, 4229].

1.D. Costal2 (Cos2, named for anterior part of the wing that duplicates [642, 1596, 3959, 4642]) is a 1201-a.a. protein that has conserved motifs of the kinesin heavy-chain (KHC) motor proteins [3612, 3976] – including domains for microtubule-binding, ATP-binding, and homodimer formation (heptad repeats). Like KHC, Cos2 binds microtubules but, unlike KHC, binding is aborted not by ATP but by receipt of a Hh signal [3612, 3976]. Genetically, cos2 acts upstream of ci [1264, 3976, 4539]. Cos2 directly binds Ci [4068, 4536] and persists in binding Ci even after microtubules dissolve [3976], so its function may be to simply

1.F. Suppressor of fused (Su(fu)) is a 468-a.a. protein whose only obvious motif is a PEST sequence [3368], although even that motif may be dispensable for the

APPENDIX SIX. SIGNAL TRANSDUCTION PATHWAYS

protein’s function [1015]. Su(fu) was thought to serve as a bridge between Fu and Ci [2906], but Fu and Ci directly bind each other [4068]. The rigid Fu:Su(fu) stoichiometry explains the dose sensitivity of fu relative to Su(fu) alleles [3368, 3459, 4280] as well as similar titrations with cos2 [3461]. Like Fu, Su(fu) exerts effects on Ci-155 levels that make no sense unless an unstable “matured” form of Ci-155 exists [3177]. Some sort of maturation must occur because an uncleavable Ci-155 construct cannot enter the nucleus [2833, 4536] or upregulate ptc [2832] without the Hh pathway being active. Dose-sensitive interactions of Su(fu) with ci suggest that Su(fu) physically blocks Ci maturation [3177]. Amazingly, Fu and Su(fu) can both be removed from the transduction machinery without altering the wild-type phenotype [78, 3459], so Ci-155 cannot require catalytic alteration by Fu [2832]. Ci’s maturation might just involve (1) avoiding PKA-mediated phosphorylation [749, 4538] or (2) allowing phosphorylation but then escaping proteolysis by rapidly entering the nucleus [4540]. In either case, maturation appears to require that all Su(fu) be “cleaned off” from Ci before Ci enters the nucleus [2833, 4068, 4536]. Otherwise, the Su(fu)-bound Ci complex cannot activate transcription at high levels. Oddly, Cos2 (once thought to only antagonize Hh signaling) assists this cleansing (hence abetting Hh) [4536] in some way that does not entail competitive binding (because Cos2 and Su(fu) bind different parts of the Ci protein).

1.G. PKA is cAMP-dependent Protein kinase A [3920], and DC0 (0 = zero, not the letter O) is its catalytic subunit [2118, 2403, 2822]. LOF alleles of DC0 mimic hhGOF phenotypes in discs [2058, 2059, 2491, 2533, 3238], plus Hh’s effect on Ci [2072] (but see [2214]). Double mutants show that DC0 acts downstream of smo [754, 755, 4441], but no direct Smo-PKA link is known [751, 1554, 1701, 2214]. PKA inhibits Hh signaling by phosphorylating Ci [731, 749, 751, 3466, 4539] (in addition to other kinases [731]), although, ironically, PKA may also be needed for Ci to mature into its active form [4540] (but see [4538]). Phosphorylation marks Ci for proteolytic cleavage (and inactivation) by the ubiquitin pathway as gated by Slimb [2668, 4538]. Hh signaling reduces Ci phosphorylation [731], but this effect could be mediated by a phosphatase acting in parallel with PKA [731, 4538]. A PKAanchoring protein has been identiﬁed in discs [1704, 4470] that could (1) put PKA near Smo [3685] or (2) attach PKA to the same microtubule sites as the Cos2-Fu-Su(fu)Ci complex [3685]. The latter idea seems to obviate the need to postulate ≥2 active Ci isoforms and would explain why Cos2 and PKA do not ﬁt a linear pathway

287

To wit, cos2LOF mutations might block Hh signaling by undocking the Fu-Su(fu)-Ci remnant from a separately anchored PKA. Similar reasoning might also explain why deleting both Su(fu) and Fu yields a wildtype phenotype [1974]. The ability of excess Ci to bypass inhibition by PKA [65] is also consistent with the notion that PKA cannot access Ci freely via 2nd-order kinetics. The basic idea can be visualized as a “CyclopsScenario,” wherein a Cyclops (PKA) cannot eat (phosphorylate and hence proteolyze) Odysseus (Ci) unless he is a captive (held by Cos2-Fu-Su(fu) handcuffs) in a cave (microtubule network) [2833, 4537]. (PKA’s phosphorylation of Ci per se has no impact on nuclear import [4540].) This scenario could be true even if ≥2 active Ci isoforms exist. Scaffold-dependent phosphorylation, as seen here, may be used elsewhere to insulate signaling pathways from one another [3454]. N.B.: One snag in trying to analyze this system is that each cog probably binds several other cogs [2906, 4068], thus confounding any attempt to chart a linear chain of epistases via genetics [2479]. A similar mess has been encountered with a protein complex (Eyes absent, Dachshund, Sine oculis) that switches on eye development (cf. Fig. 7.3 and Ch. 8) [553, 743, 3387]. [3176, 4538].

1.H. Slimb (“Supernumerary limb”) is a 510-a.a. protein [2060, 4279] whose F-domain and WD40 motifs allow it to form a bridge between ubiquitin ligases and proteins that have been tagged (e.g., by phosphorylation) for ubiquitination [791, 2668, 4031]. Ci appears to be such a protein [4538], although its proteasome-dependent cleavage [731] appears to occur sans ubiquitination [731] or further degradation [1974]. Epistasis experiments place slimb downstream of, or parallel to, DC0, and upstream of ci [4538]. Slimb is also used by the Wg pathway (see below) [2060, 2856, 4279]. The rationale for Slimb’s rapid turnover of activators like Ci and Arm is probably that it allows quick responses [4294]. Such speed is undoubtedly useful in the eye where ommatidia are spawned by a “forest ﬁre” that ﬂashes across the disc, although it may be useless in leg and wing discs where cell states change at a more languid pace. 1.I. Cubitus interruptus is named for a gap in the cubital (4th) wing vein [4092, 4094, 4106, 4107]. Formerly called CiD [3818], Ci is a 1396-a.a. protein [2832] in the Gli family of transcription factors [3686] that bind the core consensus TGGGTGGTC [2980, 4503] (but see [154]). In thoracic discs, ci mRNA is found only in the A compartment, where it is uniform [1135, 2967, 3177, 3818, 3991]. Full-length Ci (155 kD) predominates near the A/P boundary [65, 2967, 3177, 3991] in a band ∼7–9 cells wide [1828, 4539, 4540]. More anteriorly

288

(beyond the range of Hh), Ci is cleaved (somewhere in the a.a. #650–700 interval [155]; numbering as per [2832]) through Slimb-mediated [4538], proteasome-dependent [4539] proteolysis to yield a 75-kD N-terminal fragment [155] (Fig. 5.7). Ci-155 and Ci-75 function respectively as transcriptional activator vs. repressor [155, 731, 2832]. Ci155 is mainly cytoplasmic due to C-terminal sections (including a.a. #703–836) that counteract an NLS (a.a. #596–614) in Ci-75 [4539]. Because Ci’s DNA-binding domain of 5 tandem zinc ﬁngers (Cys2 His2 class; a.a. #453– 603) [65, 3194, 3292] is N-terminal to the cleavage site, both Ci-155 and Ci-75 can bind DNA and hence compete for target promoters [154, 751, 2980, 3685]. However, Ci-155 and Ci-75 have distinct (overlapping) sets of target genes [2832], so other factors must edit their speciﬁcities [2980] (e.g., see [1686, 1827]). Domains that are C-terminal to the cleavage site (and hence cropped from Ci-75) include (1) a nuclear export signal (a.a. #675–860) [731, 4539] initially thought to be a cytoplasmic tether site [155], (2) 5 PKA phosphorylation sites [3466, 4539], (3) an acidic section that activates transcription [65, 1828], and (4) a binding domain (a.a. #1020–1160) that recruits the co-activator dCBP [54, 752]. Unlike Ci-155, Ci-75 is a repressor because it lacks the latter two domains, while retaining an alanine-rich “repressor” section (a.a. #1–453) [65]. Until recently, the genetics of ci was muddled by the neomorphic ci D allele [154, 2832, 3191, 3818]. That mutation turns out to involve a quirky fusion of ci with its neighboring gene pan [3831, 4502]. In what may be the ultimate example of crossedwire “short circuiting” (Inv D is a contender [3934]), the chimeric CiD protein (Ci-Pan) regulates Hh target genes (via Ci’s zinc ﬁngers) in response to Wg (via Pan’s Armbinding domain; cf. Wg pathway). Why on earth the effector genes for the Hh and Wg pathways (ci and pan) should abut one other (∼10 kb apart) in the wild-type ﬂy [519, 1088, 4439] is bafﬂing [3831]. It is hard to believe that this juxtaposition is an evolutionary accident given the proximity of Ci and Pan binding sites at jointly regulated promoters [3386] (cf. another peculiar case [671]). From a developmental standpoint, the riddle is why ci D ’s effects are so mild in heterozygous condition. Ci may not be the only transcriptional effector for Hh [2494] (but see [4540]).

1.J. Drosophila CREB-binding protein (dCBP, where the “CREB” bZip transcription factor is the cAMP response element binding protein [88, 3879, 4393, 4817]) is a gigantic 3190-a.a. (332-kD) protein (a.k.a. Nejire) [54] that binds Ci (as a co-activator) and increases (by ∼60fold) Ci’s activation of target gene transcription [54]. The ability of dCBP to also bind phosphorylated dCREB2 [54]

APPENDIX SIX. SIGNAL TRANSDUCTION PATHWAYS

means that PKA might have an indirect effect on Ci (besides its direct effect [3466]). Namely, by phosphorylating dCREB2, PKA could cause it to “steal” dCBP from Ci if the amount of dCBP is limiting [54, 100, 2023]). CBP proteins stimulate transcription [3279] by acetylating histones [202, 1553, 2726, 3174, 3334] (as do their associated factors [2489, 2911, 3998, 4518, 4718]), and this is presumably the role of dCBP as a co-activator in the Hh pathway. In the Wg pathway, however, dCBP plays an inhibitory role (see below) by acetylating a transcription factor (Pan) so that it can no longer bind a co-activator (Arm) [4533]. This involvement of dCBP in multiple pathways is consistent with CBP’s ability to integrate diverse inputs [705, 1506, 1553, 2023, 2124] by dint of its huge size [2024].

1.K. Target genes include the following. Some of these links have been conﬁrmed in cell culture [65, 731, 4503], although in most cases it is unclear whether the effect is direct or indirect. A likely target in the wing pouch (turned off by Hh) is Egfr [1643, 4604], and an indirect target (via en) is polyhomeotic [2728, 2729]. hh itself, which is kept off in A cells of leg and 1. Hh wing discs by Ci-75 (when no Hh signal is received), although hh is unresponsive to Ci-155 [2832]. An autoregulatory loop of this sort also operates in the developing eye [1786, 3238]. 2. Hh ptc, whose low-level transcription in A cells is upregulated by Hh near the A/P boundary [2834] via binding of Ci-155 to the ptc promoter [731] but whose lower level in remaining A cells is not due to Ci-75 [2832] . 3. Hh en, which is turned on by Ci-155 in A cells near the A/P boundary [2834] during late 3rd instar [78] (cf. similar effects in ptcLOF A clones [3177] ). This incursion of the en-on state into the A compartment is stopped by fuLOF [1537, 3739]. 4. Hh dpp, which is turned on by Ci-155 along the A/P border in wing discs [65, 754] by binding of Ci155 to a dpp enhancer [2980] , whereas Ci-75 reduces dpp’s basal (default) transcription level to zero beyond the range of Hh [2834] . Likewise, dpp is turned on dorsally in leg discs [3739] , turned on ventrally in antennal anlagen [697] , and turned on in the MF of the eye disc before [406] and during [4387] retinal differentiation except at the MF tips [406] . 5. Hh tkv near the A/P border in wing discs [4251] via master of thickveins (mtv): Hh mtv tkv [1327]. 6. Hh vein (as a direct target) in the wing pouch [4604], as well as in the dorsal head and MF [80].

APPENDIX SIX. SIGNAL TRANSDUCTION PATHWAYS

7. Hh knot (a.k.a. collier) in the wing pouch [2894] (cf. roadkill [4502]). 8. Hh blistered (via Knot [4479]) in the wing pouch [3145]. 9. Hh {araucan and caupolican} in the wing pouch [2993] IF a high level of Dpp is also present [1537]. 10. Hh wg ventrally in leg discs [231, 2466], dorsally in antennal anlagen [697] , and anteriorly in the eye rudiment [1781] , but Hh wg in the dorsal head [3664]. 11. Hh hairy anterior to the A/P boundary in leg discs [1778] and anterior to the MF in eye discs [4387] , but Hh hairy within the MF itself (a threshold effect?) [1616]. 12. Hh atonal in the MF during its progression [725] and at its inception [406], but high levels of Hh atonal at the MF’s rear [1077]. A “Hh atonal” link also operates in the developing antenna [2055] and larval eye organ [4216], but in the latter case neither Fu nor Ci is needed for Hh transduction. 13. Hh orthodenticle in the eye disc [3664]. 14. Hh glass in the developing eye [2632]. 15. Hh rough in the developing eye [1077]. 16. Hh scabrous in the developing eye [3238] . 17. Hh shortsighted ahead of the MF in the eye disc [4388].

1.L. Summary of the basic chain: Hh Ptc Smo Fu Ci {target genes}. In the absence of Hh, Ptc keeps the pathway off by repressing Smo. Binding of Hh to Ptc alters Ptc so that Smo is freed to (1) stimulate Fu, which somehow enables Ci to enter the nucleus and activate target genes; (2) liberate the Ci-Cos2Fu-Su(fu) complex from its microtubule tether, which also helps release Ci for nuclear transit; and (3) prevent PKA from inactivating Ci by phosphorylation and subsequent cleavage. How Hh represses target genes is unknown. 2. Decapentaplegic signaling pathway [2733] (see [3498] for a primer.) Aside from the components below, other candidates include crossveinless 2 [854], lilliputian [3097, 4195], Merlin [2776], expanded [2776], shortsighted [4388], vrille [1434], p38 mitogen-activated protein kinase [13], and unidentiﬁed genes at 54F–55A and 66B–C [3114] and elsewhere [1939, 3034]. Based on what is known about the vertebrate TGF-β pathway, (1) Protein Phosphatase 2A is likely downstream of (and phosphorylated by) Tkv [1627], and (2) microtubules may sequester Mad [1084]. Also, vertebrate Mad is escorted to the receptor complex (≈ Punt-Tkv) by SARA (Smad Anchor for Receptor

289

Activation) [138, 3221, 3897, 4403], and ﬂies may use the same trick because they have an ortholog (dSara) [288, 1172]. Certain components of the Dpp pathway are replaced by others during embryogenesis (e.g., see [88, 2990, 3586]), oogenesis (e.g., see [4703]), and patterning of the larval midgut (e.g., see [4203, 4226]).

2.A. Decapentaplegic (Dpp) is so named because at least 15 (“decapenta-”) of the 19 imaginal discs are incapacitated (-plegic) by dpp mutations [4033]. Despite the presence of 5 differently spliced dpp transcripts [4056], there is only one Dpp protein [3096]. Like other members of the TGF-β family, Dpp (BMP4 subfamily [2733, 2735]) is translated as a precursor (588 a.a.) [3224] and cleaved into (1) an N-terminal piece (DppN , a.a. #1–456) that fosters dimerization and secretion and (2) a C-terminal ligand (DppC , a.a. #457–588) for intercellular signaling [1427, 3244]. The ligand has a consensus N-glycosylation site [1427, 3585] and 7 conserved cysteines [3096]. Six of the cysteines join pairwise in disulﬁde bridges to weave the TGF-β “knot” [2734], and the 7th links the Dpp monomers [4608]. The protease that cleaves the precursor was thought to be a BMP1 relative [2696] but is probably a subtilisin cousin [1232]. Aside from Dpp, 5 other TGF-β family members have been characterized: dActivin [2369, 3222], Glass bottom boat (Gbb, a.k.a. 60A) [1068, 4614], Maverick [3110], Myoglianin [2578], and Screw [109] (cf. EST DS07149 [3498] and [3674]). Among these paralogs, only Gbb is known to affect Dpp signaling in discs, but its inﬂuence is slight and limited mainly to wing veins [1674, 2203, 4610]. Historically, dpp genetics was complicated because most dpp mutations disjoin cis-enhancers from the coding region [1426, 4056, 4608] (cf. the achaete-scute Complex, Ch. 3). The locus spans ∼55 kb [3098, 4056] and is organized into (1) an ∼8 kb “Hin” (Haplo-insufﬁcient) coding section [1428, 1987] with enhancers for embryonic expression [1872, 2003, 3833]; (2) a 5 “Shortvein” region where mutations cause defects in wing veins, larval midgut, and other structures [1952, 2738, 3850]; and (3) a 3 “Disk” region where enhancers drive expression in various parts of discs [347, 2739, 4033]. 2.B. Punt (a.k.a. Activin Receptor Type II; name based on embryo phenotype) is a transmembrane (1-pass) receptor (516 a.a.) with a cysteine-rich extracellular domain and an intracellular kinase domain [772, 2495, 3668]. Punt appears to be the only “type II” receptor in D. melanogaster [3932], although there are 3 “type I” receptors (Thick veins, Saxophone, and Baboon) [511, 2495, 4754]. Type-I and -II proteins form hetero-oligomeric receptor complexes in ﬂies and vertebrates [4267, 4472, 4585, 4752] – a mix-and-match strategy that accommodates diverse

290

ligands [2732]. Both kinds of proteins are serine-threonine kinases (type II are constitutive; type I are ligand dependent) [4267, 4753], although their catalytic domains resemble receptor tyrosine kinases (RTKs) more than S-T kinases [1711, 1953], so TGF-β receptors may have evolved from RTKs or vice versa [3221]. Indeed, type-II receptors were recently found to naturally autophosphorylate on tyrosines [2418] as well as serines [2619], and like RTKs they assemble into a complex via ligand binding [505, 2731, 4753]. Dpp dimers bind type-I (Tkv) and type-II (Punt) proteins cooperatively (vs. sequentially) [2495, 2733, 3328, 4609], and there are ≥2 Punt monomers in the complex [3932].

2.C. Thick veins (Tkv, named for its LOF effect on wing veins [980, 995, 3328, 4189, 4271]) is a type-I transmembrane (1pass) receptor (563 a.a.) [512, 3073, 3328] with a cysteine-rich extracellular domain, an intracellular kinase domain, and a juxtamembrane GS (Glycine-Serine-rich) domain that is presumably phosphorylated by Punt in the presence of Dpp [1874]. After it is activated (phosphorylated) by Punt, Tkv puts phosphates on serines in Mad’s Cterminal SSxS tail [1983], and Mad then relays the signal to the nucleus. Saxophone (Sax, 570 a.a.) is another typeI receptor [512, 3073, 3328, 4771], but its LOF and GOF effects (mainly on wing veins) are meager [3972]. Sax does not normally serve as a Dpp receptor in discs [1674, 3109, 3498], despite initial suspicions that it transduces Dpp like Tkv [512, 3073, 3668, 3972, 4609]. Sax’s ability to boost the Tkv signal [3972] must therefore occur indirectly by downstream cross-talk between Dpp and a TGF-β ligand other than Dpp [3406] – possibly Gbb [3498]. 2.D. Dally (Division abnormally delayed) is a putative cell-surface proteoglycan (626 a.a.) [3038] with motifs diagnostic of the “GRIP” subfamily (Glypican-Related Integral-membrane Proteoglycan): (1) an N-terminal signal peptide, (2) a hydrophobic C-terminus needed for glypican-type anchoring to the outer leaﬂet of the membrane [966], (3) consensus sequences for glycosaminoglycan links, and (4) 14 conserved cysteines of vertebrate GRIPs (cf. type-III “receptors” in the TGF-β family [139]). LOF alleles of dally have mild dpp-like effects on eye size and wing venation [2556, 3038] (cf. similar vein defects in sugarlessLOF [1673]), and they enhance dppLOF phenotypes in double mutants [2004]. The ability of dallyLOF to reduce expression of dpp target genes (omb and spalt) without affecting dpp expression itself suggests that dally acts downstream of dpp, but the ability of extra doses of dpp+ to rescue dallyLOF defects argues that dally acts upstream of dpp [2004]. The latter is more consistent with Dally’s supposed role (based on molecular features) as

APPENDIX SIX. SIGNAL TRANSDUCTION PATHWAYS

a co-receptor for Dpp and Wg on receiving cells [2004, 2556, 3345].

2.E. Mothers against dpp is a 455-a.a. transcription factor [2217, 3852]. The gene name is capitalized because of a dominant maternal (hence “Mothers”) effect that MadLOF mutations have on the lethality of dppLOF embryos [3499, 3852]. The name is a pun on the American association “Mothers Against Drunk Driving,” but “against Dpp” is misleading because Mad actually assists (rather than hinders) signaling (MadLOF enhances dppLOF ). Its abbreviation “Mad” is also unfortunate because it can be confused with (1) vertebrate Mad (an unrelated bHLH-Zip transcription factor [1594]) or its ﬂy ortholog dMad [2596, 4820], (2) mad (the unrelated ﬂy gene many abnormal discs) [2561], or (3) the MADS box (an unrelated DNA-binding domain). Genetically, Mad functions downstream of dpp [3094] and tkv [3095]. Mad is the founder of the Smad family of regulators [137, 138, 174, 4638], which contains 3 subgroups [1815, 2332, 2736]: (1) pathwayspeciﬁc Smads (e.g., Mad and dSmad2 [511]), (2) common mediators (e.g., Medea), and (3) inhibitory Smads (e.g., Dad). Smad proteins share one or two homology domains with Mad (separated by a proline-rich linker). The MH1 domain binds DNA [2217, 3898], whereas MH2 binds the receptor [756, 2579]. MH2 also mediates oligomerization [2380] and activates transcription [2568] (autonomously and by recruiting CBP [138, 2332, 4534]). Oddly, MH1 inhibits the CBP-binding ability of MH2 [4534], while MH2 inhibits the DNA-binding ability of MH1 [2217]. Evidently, phosphorylation cures Mad’s self-defeatism by reshaping the molecule [1815, 2332, 3498, 4751]. Pathway-speciﬁc Smads end C-terminally in SSxS (where “x” is variable) and cannot transmit signals unless the serines bear phosphates (but see [2380]). Phosphorylation of Mad’s SSxS tail by Tkv [1983, 3095] triggers (1) formation of Mad homo-oligomers, (2) formation of Mad-Medea hetero-oligomers, and (3) entry of the latter complexes into the nucleus [959, 1983, 4703], although Mad can enter sans Medea [959, 4703]. Mad binds the consensus nucleotide sequence GCCGnCGC (where “n” is variable) [2217]. Mad is expressed ubiquitously in wing discs (and presumably other discs) [3095]. 2.F. Medea is a 745-a.a. protein [959, 1938, 1983, 4703] (but see [4782]) that belongs to the “common mediator” subgroup of the Smad family [1815]. Like other subgroup members, Medea has MH1 and MH2 (Mad homology) domains and lacks a C-terminal SSxS [1983]. It also has an opa motif that is lacking in its human ortholog Smad4 [1938]. Medea was found in the same screen as Mad [3499] and was likewise named for its maternal enhancement of dppLOF

APPENDIX SIX. SIGNAL TRANSDUCTION PATHWAYS

embryos’ lethality. (In Greek mythology, Medea was a sorceress who killed her children.) Genetically, Medea acts downstream of dpp [4703] and tkv [1938] (but see [959]). Medea binds Mad (via Medea’s MH2 domain) only when Mad’s SSxS serines are phosphorylated [959, 1983, 4703], and this binding is needed for Medea to enter the nucleus [4703]. In the nucleus, Medea is a partner for Mad in DNA binding [4703, 4782], although the binding complex’s constitution and conﬁguration are uncertain [1027, 4638]. In contrast to Mad, Dpp signaling does not involve phosphorylation of Medea, and endogenously high levels of Dpp can activate target genes (omb in wings) without Medea [4703].

2.G. Daughters against dpp (Dad) is a 568-a.a. Smad protein [4404] whose name parodies Mad. Like other members of the inhibitor subgroup, Dad has MH2 but lacks MH1 and SSxS [3498, 4637]. Genetically, Dad acts downstream of dpp but upstream of omb, whose expression it represses [4404]. This repression is due to Dad’s ability to prevent Mad phosphorylation by Tkv [1983] – a blockage that seems steric and stoichiometric because (1) Dad competes with Mad for binding to Tkv (Dad binds more tightly) [1983] and (2) overgrowth caused by excess Mad can be rescued to wild-type by overexpressing Dad coincidently [4404]. Dpp signaling activates Dad transcription, which represses Dpp signaling [4404] – ergo, a negative feedback loop. This loop should make cells in the Dad zone (near the Dpp stripe; Fig. 6.2) perceive the Dpp gradient as ﬂatter than it really is [4637, 4638]. How much the perceived and actual slopes differ is hard to guess without knowing afﬁnities and amounts of all the relevant players. The only certainty is that Dad can’t be buffering Dpp’s signal too strongly because in that case Dpp could not act as a morphogen (cf. Fig. 6.3). 2.H. Brinker (Brk) is a 704-a.a. transcription factor with weak homeodomain homology, 3 opa repeats, 3 possible NLSs, several copies of a motif that recruits the dCtBP co-repressor [619, 2049, 2867], and an FKPY motif that recruits the Groucho co-repressor [4866]. Brk binds (and needs) Groucho more strongly than dCtBP [4866]. Its Nterminal helix-turn-helix (homeo-like) domain binds a speciﬁc DNA sequence at omb [3978] and other loci [2416, 3696, 4866]. Because brknull somatic clones express Dpp target genes (Dad, omb, spalt) in areas where they are normally off [2049, 2867], Brk’s normal duty may be to keep these genes off. The ability of brknull to turn on omb and spalt in the absence of Dpp signaling (in brknull clones that are also Mad null or tkv null ) argues that Dpp acts via Brk – i.e., Dpp brk {omb and spalt} [619, 2049, 2050].

291

However, spalt upregulation does not attain in situ levels (nor do structures attain a “Dpp peak” fate as fully as in tkvGOF clones) [619, 2049], so Brk may act partly in parallel with Dpp [2049]. Additional evidence for downstream (in-series) placement of Brk is the ability of brkLOF to restore tiny dppLOF discs to normal size [619], although here too spalt expression is abnormal. Dpp represses brk transcription [619, 2049, 2867], so Brk forms a gradient that overlaps and opposes the Dpp gradient. (Its expression complements Dad’s [2867]; Fig. 6.2.) Indeed, Brk may help cells distinguish Dpp levels at the shallow end of the Dpp gradient [619, 2049]: if Mad overcomes Brk at a target promoter, then the gene should switch on, whereas if Brk wins, then the gene should turn off [619, 2049, 2233]. Thresholds may thus be sharpened [3347, 3406]. Brk cannot be regulating itself because brknull clones express a brk enhancer-trapped reporter in a normal pattern [2867]. Rather, brk is regulated by Schnurri (see below) [1749, 2727]. Brk may also participate in other pathways [2397].

2.I. DrosophilaCREB-bindingprotein (dCBP) is a huge 3190-a.a. protein (cf. Hh pathway) [54] that activates transcription when recruited to DNA by DNA-binding proteins [202, 2726, 3174]. Ci is one such recruiter, and so is Mad [127, 4534]. This co-activator role for dCBP in Dpp signaling agrees with the genetic placement of dCBP downstream of sax [4534]. 2.J. Schnurri (Shn) is a 2528-a.a. protein with 7 (or 8 [4059]) zinc ﬁngers, an acidic (activator?) domain, and multiple opa motifs [108]. Until recently, it was unclear where Shn ﬁt with other components of the pathway [3223, 3498], especially because some of its interactions are allele speciﬁc [753]. We now know that Shn binds DNA and physically interacts with Mad as a cofactor to affect transcription of target genes [944]. Its chief target appears to be brinker (Shn brk) [1749, 2727] (but see [4372]). Shn is needed for Dpp signaling in wing discs (for A-P patterning and vein formation) [4373] as well as during embryogenesis and midgut formation [569, 1622, 4059]. Shn (with Punt) also transduces signals from a ligand other than Dpp during spermatogenesis [2751]. 2.K. Target genes include: dpp itself in an analog manner by a Tkv1. Dpp mediated autocrine loop until a set point is reached (≈ thermostat) in wing discs [1674]. However, Dpp dpp at the tips of the MF in the developing eye [4387] and at the anterior margin [3388]. Given Dpp’s ability to diffuse, the latter loop should incite the dppon state to spread like wildﬁre. Spreading is indeed

292

2.

3.

4. 5.

6. 7. 8.

9. 10.

11. 12. 13. 14. 15. 16.

17.

18.

19.

APPENDIX SIX. SIGNAL TRANSDUCTION PATHWAYS

seen wherever the link works [724], but it is normally damped by “Wg dpp” [4387] . Dpp tkv in prospective veins of pupal wing discs [980] but not earlier (in the pouch), except at unnaturally high doses (cf. Ch. 6) [1674, 2457]. Dpp brk within ∼20 cells from the Dpp stripe in wing discs [619, 2049, 2867] and leg discs [2049]. Repression of brk (in the wing at least) is mediated by Shn [2727] : Dpp shn brk. Dpp Dad in the same zone as brk [4404]. Dpp optomotor-blind (omb) in the leg disc [2049] , in the eye disc [3978], and in the same zone of the wing disc as Dad except for the notum [619] . The leg and eye cis-enhancers reside in omb’s second intron, whereas the wing enhancer is in a 284-b.p. piece that lies 27 kb upstream of the transcription start site [3978]. Dpp spalt in the wing pouch in a narrower band than omb [619] . Dpp vestigial in the wing pouch [619] . Dpp {araucan and caupolican} in the wing pouch [1537] IF Hh is also present (and transduced to Ci-155). Dpp homothorax in the wing pouch [157] via vestigial (at least in part). Dpp wg in leg discs [487, 2059, 2082, 2954, 4277] and eye discs [1780, 4387] , but Dpp wg in the notal portion of the wing disc [3763, 3764, 4369] (but see [4368]). Dpp pannier in the notal portion of the wing disc [3764, 4369]. Dpp u-shaped in the notal portion of the wing disc [3764, 4369]. Dpp {achaete and scute} in the notal portion of the wing disc [4368] via spalt (at least in part) [987]. Dpp BarH1 in the notal portion of the wing disc [3763]. Dpp aristaless in leg and wing discs (at a certain threshold) IF Wg is also present [643] . Dpp dachshund in leg discs (at a certain threshold) IF Wg is also present [2456], although this same link works without Wg in the eye disc (before initiation of the MF) [930]. Dpp Distal-less in leg discs (at a certain threshold) IF Wg is also present [2456] , and the same is true for antennal anlagen [1037]. Dpp hairy in leg discs IF Hh is also present [1778]. The same link operates in eye discs but does not require Hh [1616]. Dpp eyes absent in eye discs before initiation of the MF [930].

sine oculis in eye discs before initiation of 20. Dpp the MF [930]. 21. Dpp hh when dpp is misexpressed at the anterior edge of the eye rudiment [406]. 22. Dpp eyeless in eye discs IF Wg is also present [2465].

2.L. Summary of the basic chain: Dpp Punt/Tkv Mad {target genes}. Binding of Dpp to Punt/Tkv causes (1) phosphorylation of Mad by Tkv, (2) entry of activated Mad (complexed with Medea) into the nucleus, and (3) stimulation of target gene transcription by dCBP that is recruited by Mad. How Dpp represses target genes (aside from brk) is unknown. 3. Wingless signaling pathway [598, 2720, 4708] (see [3919] for a synopsis and [3411] for mechanics). Aside from the components listed below, fringe connection ( frc) [3855], gammy legs (gam) [599], Lobe [758], naked [1603], nemo [304, 2872, 4475], skinhead [3371], split ends (spen) [2554], tartaruga (trt) [2290], and at least 6 other unidentiﬁed genes have been implicated [891, 2984], although some of them may affect the pathway indirectly via Arm’s secondary (nonsignaling) role in cell junctions [3873]. For example, arc encodes a PDZ-domain protein that co-localizes with Arm in junctions [2571], but its LOF phenotype (curved wings) does not reveal any obvious involvement with Wg transduction per se. The Bright-family protein Eyelid (a.k.a. Osa [4468]) inhibits Wg signaling [4389], but whether it belongs in the pathway is unclear. The zinc-ﬁnger protein Combgap acts downstream of dAxin to transduce Wg in optic lobes [4017], but it transduces Hh signals in leg, wing, and eye discs [621, 4217]. “Notum” antagonizes Wg (at high Wg doses) in a negative feedback loop (Wg Notum Wg signaling) [1494] that resembles Dad’s effect in the Dpp pathway (Dpp Dad Dpp signaling) [4404]. One heretical result in need of further investigation is the apparent ability of Notch to respond to Wg without being cleaved from the membrane [4601, 4603], as well as the attendant notion that Notch can serve as a receptor for Wg [726]. 3.A. Wingless (Wg) is a secreted protein [920, 1541, 4442, 4455], which, in nascent form (468 a.a.), contains a signal peptide, 23 conserved Cys residues, and one N-linked glycosylation site [175, 590, 3599]. It is the best characterized of the ﬂy’s four analyzed Wnt genes [1150, 1277, 1592, 2315, 3700] – one of which (DWnt-4) antagonizes Wg signaling [557, 1482]. Three more Wnts exist in the genome, equaling a total of seven [3674]. Wg can be secreted with or without glycosylation [3561], and both forms are active as ligands, as are nonsecretable Wnt constructs that are

APPENDIX SIX. SIGNAL TRANSDUCTION PATHWAYS

tethered to the cell surface [3269]. The degree of glycosylation affects Wg’s ability to bind glycosaminoglycans of the extracellular matrix [3561]. That binding is apparently needed for signaling in vivo because removal of Porcupine (Porc) – which helps glycosylate Wg before secretion [2110] – causes wgLOF -like phenotypes.

3.B. Drosophila Frizzled2 (Dfz2, named for disturbances in bristle polarity) is a 694-a.a. (7-pass) transmembrane receptor for Wg [310, 595, 4867]. Dfz2 is one of 4 Frizzled-family proteins identiﬁed in D. melanogaster [422, 1971, 3674, 3762, 3977]. The only other well-studied member is Frizzled (Fz) [26, 2088, 2325, 3264], which functions in a “planar cell polarity” (PCP) pathway [2326, 2884, 3912, 4702] that controls the orientation of bristles [1449, 1640, 1810], hairs [22, 846, 4430, 4739, 4839], and photoreceptor clusters [1639, 3912, 4170, 4365, 4873]. The unknown ligand for the PCP pathway is probably another Wnt [310, 421, 2781, 3912, 4573]. The Wg and PCP pathways were originally thought to use distinct receptors (i.e., Dfz2 vs. Fz) [422, 3912, 4867], despite (1) their shared use of Dsh (see 3.E below) [3316], (2) pathway interactions in GOF studies [4365], and (3) redundancy of Fz and Dfz2 under some LOF conditions [311, 312, 2187, 2984] (but see [595, 4867]). However, this idea seemed untenable when a Dfz2null allele became available in 1999, and Fz and Dfz2 were shown to be interchangeable – thus afﬁrming their redundancy [734] (but see [421, 2899]). Nevertheless, in 2000, Wg was indeed found to have a 10-fold greater afﬁnity for Dfz2 than for Fz [3691]. Conceivably, the elusive ligand for planar polarity prefers Fz because accessory factors form a co-receptor complex with it but not with Dfz2 [421, 734]. Transduction of Wg stops in the absence of both Fz and Dfz2 (arguing against other receptors), except in the notum where a third Wnt may preside [678]. Like all Frizzled family members, Dfz2 has a conserved ∼120-a.a. extracellular domain (with 10 cysteines at ﬁxed sites) and an idiosyncratic cytoplasmic domain [3203, 4544, 4784], wherein lies part of its speciﬁcity [421, 3691]. It is unclear how Dfz2 physically transduces the Wg signal [310, 421, 598, 4708] and relays it to Dsh [422, 2883, 3130, 3919, 4795]. Receptor dimerization is probably involved given what is known about ﬁbroblast growth factor (FGF) [3322, 3403, 3404], which resembles Wg in its use of glycosaminoglycans (GAGs) as co-receptors [3561, 3784]. Indeed, FGF [2555] and Wg receptors [2556, 4400] both rely on Dally [3038], apparently for this purpose. Relay of signal from Dfz2 to Dsh may involve Wg-dependent recruitment of Dsh to Dfz2 (via binding of Dfz2’s C-terminus by Dsh’s PDZ domain) and subsequent activation of Dsh by a membrane-linked kinase (Casein kinase 2?) [3344].

293

3.C. Dally (Division abnormally delayed) is a putative cell-surface proteoglycan (626 a.a.) [3038, 3345] (see Dpp pathway above). Dally’s GAG moiety is synthesized via Sugarless (Sgl, a UDP-glucose dehydrogenase) [338, 921, 1664, 1673] and Sulfateless (Sﬂ, a heparan sulfate deacetylase/sulphotransferase) [2556, 4400]. Indeed, LOF mutations in all three genes (dally, sgl, and sﬂ ) cause wgLOF like phenotypes. Dally is probably a co-receptor for Wg (i.e., part of a complex with Wg and Dfz2) but could instead just be helping Wg reach Dfz2 by holding Wg at the membrane [2556]. In embryos, a protein resembling Dally is expressed in D-V ectodermal stripes adjacent to dally-on stripes [2204]. The gene dally-like (dlp) acts differently from dally because its GOF phenotype mimics dally’s LOF phenotype in wings [166]. Like Dally, Dlp is a glypican (765 a.a.), but instead of helping Wg reach its receptor, Dlp sequesters Wg and reduces its potency as a ligand [166]. 3.D. Arrow (Arr, named for segment polarity defects [3152]) is a one-pass transmembrane protein (1678 a.a.) with a signal peptide, 4 EGF-like repeats, and 3 “LDLR” (low-density lipoprotein receptor) repeats [4570]. It acts downstream of Wg and upstream of Dsh [3607, 4570, 4573]. Unlike dally, sgl, and sﬂ, the LOF effects of arr cannot be suppressed by excess Wg, so Arr is probably not functioning in presenting the Wg signal [4570]. The identity of arr null and wg null phenotypes argues that arr is squarely in the transduction chain (not in a parallel or redundant pathway) [4570], but exactly where and how Arr might interact with Dfz2 is unclear [4571]. The best guess at present is that Arr and Dfz2 cooperate to form a receptor complex [270, 3241, 4242, 4570]. 3.E. Dishevelled (Dsh, named for disturbances in bristle polarity) is a 623-a.a. cytoplasmic protein [4795] with three conserved domains: PDZ (a scaffolding motif; cf. App. 1), DIX (Dishevelled-Axin), and DEP (DishevelledEGL-10-Pleckstrin) [422, 2262, 4278]. The DIX and PDZ domains are crucial for Wg signaling (and subcellular localization [152]), whereas the DEP and PDZ domains are involved in the PCP pathway [422, 423, 2238, 2883, 4795] (but see [152]). Downstream of the DEP-PDZ branchpoint, the PCP signal is transduced by dRac1 (GTPase) [1132, 1136, 1195], RhoA (GTPase) [4170], Misshapen (kinase) [3261], JNK ( Jun N-terminal Kinase) [423, 2781], and dJun (leucinezipper transcription factor) [4566]. Other nodes (of uncertain linkage [2883, 4173, 4566]) in the PCP chain include Dachsous [21], Expanded [367], Prickle (a.k.a. Spiny Legs [1641, 1795, 2884]), Starry Night (a.k.a. Flamingo) [704, 4430], and Van Gogh (a.k.a. Strabismus) [25, 4263, 4716]. (Apical-basal

294

polarity uses an entirely separate circuit [2983].) Dsh also affects the Notch pathway by binding Notch directly [151, 356]. Dsh is hyperphosphorylated by Casein kinase 2 in response to Wg [3149, 3683, 4365, 4676, 4795], although reports differ as to whether phosphorylation alone is sufﬁcient to relay the signal [422, 3683, 4676]. Genetically, dsh functions upstream of sgg as a negative regulator [882, 1833, 3683, 3924]. Dsh lacks an obvious catalytic domain and seems to act rather as an Axin-binding adaptor that recruits Sgginhibiting agents to the Axin-Sgg-Arm complex [3316, 3994]. Overexpression of Dsh (or Wg or Dfz2) inactivates Sgg by causing it to be serine-phosphorylated [3683], apparently via Protein kinase C [859, 3889]. Dsh and Arm both help pattern the V side of the leg, but only Arm is essential for cell viability there [2262, 4278].

3.F. Shaggy (Sgg, named for extra bristles, a.k.a. Zestewhite 3) is a serine-threonine kinase (514 a.a. for the most prevalent of the 5 isoforms) [419, 3682, 3920, 3922] homologous to vertebrate GSK-3β (glycogen synthase kinase3β) [3921, 4741]. In keeping with its role in other systems, Sgg keeps the pathway tonically off in the absence of Wg [4741]. Genetically sgg acts upstream of arm [3228, 3315, 3318, 3924, 4796], and its net effect is to reduce the pool size of free Arm by causing Arm to be phosphorylated, ubiquitinated, and degraded [3204, 3228, 3315]. Sgg can phosphorylate Arm [3683], but Sgg may not be Arm’s natural kinase [3228, 3344, 4062, 4678, 4679]. Because Sgg can phosphorylate dAxin [3683] and probably also APC, one or both of these agents might relay the Wg signal from Sgg to Arm (cf. dAxin). Indeed, Dsh-vh inhibits both of these reactions when it binds Axin [2238]. This inhibition is attributable to reduced enzymatic activity of Sgg and ultimately to serine phosphorylation of Sgg by an unidentiﬁed kinase [3683]. 3.G. E-APC (Epithelial Adenomatous Polyposis Coli; a.k.a. dAPC2) is the product of a Drosophila homolog of vertebrate APC [2775, 3468, 4832, 4833]. Whereas E-APC is expressed ubiquitously, the other known homolog – dAPC (Drosophila APC ) – appears to function only in photoreceptor cells [39, 1766]. In vertebrates, APC binds Sgg-vh, Arm-vh, and Axin [1593, 2239, 2996, 3675, 4040] and helps target Arm-vh for degradation [2239, 2914, 2997, 3304, 3307]. E-APC likewise binds Arm [2775, 4832, 4833] and seems to play a similar role [2775]. Its association with “adherens” junctions (see below) [2608] suggests that E-APC ferries Arm to Axin [322, 4374], and it may actually be the bridge (instead of dAxin) between Dsh and Sgg [3344, 4833]. Consistent with a ferrying role, E-APC has nuclear export sequences that must be intact for Arm to exit the nucleus [3650].

APPENDIX SIX. SIGNAL TRANSDUCTION PATHWAYS

3.H. Slimb (“Supernumerary limb”) is a 511-a.a. protein [2060, 4279] whose F-domain and WD40 motifs enable it to link ubiquitin ligases with speciﬁc ligase substrates [791, 2668, 4031], including Arm [2856]. The effects of slimb null are not spatially congruent with those of sgg null [2060, 4279], probably because Slimb also participates in other pathways (e.g., Hedgehog signaling above; cf. IκB degradation [4476]). 3.I. dAxin (739 a.a.) is the Drosophila homolog of vertebrate Axin [1698, 4677, 4861]. It uses a central conserved sequence to bind Arm and an N-terminal “RGS” (regulator of G-protein signaling) domain [2282] to bind dAPC (but see [4040]). Notwithstanding an initial report to the contrary [1698], dAxin also binds Sgg [3683, 4677] near dAxin’s Arm-binding site. The 80-a.a. DIX domain shared by Axin and Dsh (at their C- and N-termini, respectively) can also mediate protein binding [1919, 2238]. Indeed, vertebrate Axin is thought to be the scaffold on which a protein complex is built [598, 1182, 3316], consisting of APC, PP2A, the homologs of Dsh, Sgg, Arm, and Slimb [1919, 2238, 2523, 3309, 4495], and Casein kinase [3724]. Genetically, dAxin behaves as a negative regulator of Wg signaling upstream of arm [1698, 2465, 4677]. Given that (1) Sgg phosphorylates dAxin in Wg-stimulated cells [3683], (2) phosphoAxin has a greater afﬁnity for Arm-vh [4679] (but see [1959]), and (3) phospho-Axin keeps Arm-vh attached to the ubiquitination machinery [2241], it follows that dAxin may be relaying the Wg signal by releasing Arm when Dsh blocks Sgg [1182, 4679]. When Axin is dephosphorylated by PP2A (serine-threonine Protein Phosphatase 2A, another Axin-binding partner [1919, 3849, 4679]), Sgg dissociates from the complex and Sgg-dependent phosphorylation of Arm subsides [3683]. Thus, Arm’s release is also likely due to Slimb’s low afﬁnity for unphosphorylated Arm [1736, 2241, 4701]. 3.J. Armadillo (Arm, named for embryo segmentation defect) is an 843-a.a. protein (a 721-a.a. isoform exists in the CNS [2604]) with 12 protein-binding “arm” repeats (cf. App. 1) [892, 3320, 3598] and an acidic C-terminal region that activates transcription [892, 3146, 4439]. Arm is homologous to vertebrate β-catenin [3320, 4622]. Like its counterpart, Arm is detectable in the cytosol but enriched at cellcell adherens junctions [3306, 3314, 3320, 3597, 4745]. Adherens junctions are crucial for epithelial integrity [890, 1947, 2273, 2985, 3167, 4295] and possibly some types of signaling (e.g., Wg [2608], EGFR [1001, 1905, 4081], or Notch [184]). Within these junctions, Arm acts as a linchpin between transmembranous dE-cadherin [796, 893, 3168, 3749, 4796] (a.k.a. Shotgun [4268, 4416]) and cortical dα-catenin [3166, 3169, 3409], which in

APPENDIX SIX. SIGNAL TRANSDUCTION PATHWAYS

turn is anchored to the actin cytoskeleton [956, 1937, 2185, 3412, 4807] via dRac1 [1133], etc. [1648, 2368, 3679]. Transport of Arm to the junction depends on Presenilin [3124]. Retention of Arm at the membrane depends on PP2A [1578]. Arm’s junctional role is separable from its signaling function in the nucleus [893, 2860, 3204, 3749, 4880], although various cadherins can reduce signaling by diverting Arm into junctions [1603]. Wg signaling affects dE-cadherin transcription [4796] but not arm transcription, which is uniform in discs [3598]. Rather, Wg dictates the amount of Arm posttranslationally [273, 1345, 3318, 3320, 3597] by blocking its rapid turnover [7, 4701]. In the nucleus, Arm is a co-activator for Pan [519, 4439] and possibly for other transcription factors as well [3873]. The deployment of Arm to the cytoplasm vs. nucleus appears to reﬂect Arm’s relative afﬁnities for dAxin vs. Pan [4352].

3.K. Pangolin (Pan, named for an armadillo-like embryo segmentation defect, a.k.a. dTcf = Drosophila Tcell factor) is a 751-a.a. DNA-binding protein [519, 1088, 4439] that can bend and unwind DNA by virtue of its HMG domain [266, 691, 810, 1481] (cf. App. 1). Genetically, pan acts downstream of arm [519, 3591, 4439]. Pan binds Arm [519, 4439] when the cytoplasmic pool of Arm enlarges sufﬁciently after Wg-dependent Arm stabilization [2996, 3228, 3318, 3749]. Pan may help Arm enter the nucleus [266, 1917, 2337], although Arm can do so independently [691, 4439], apparently by using its arm motifs instead of a NLS (cf. App. 1) [1180]. Within the nucleus, the Arm-Pan complex activates target genes via (1) Pan’s binding to CCTTTGATCTT [3591, 4439] and (2) Arm’s stimulating of transcription via its C-terminus [892, 1917, 4439] (but see [2697]). In the absence of Arm, Pan uses a clever trick to keep Wg-responsive genes completely off [3147]: it binds and escorts the corepressor Groucho (Gro) into the nucleus [692, 2496], and Gro then elicits remodeling of the chromatin [737, 849]. Pan’s binding of Arm or Gro may be mutually exclusive [3147], and Arm’s binding of Pan or dE-cadherin is competitive [1593, 3202, 3204, 3749], so dE-cadherin and Gro apparently set thresholds that the level of Arm must exceed before Arm can help Pan turn on genes [849, 893, 2860]. Pan might also bind Gammy legs, which acts downstream of Arm (as a cofactor?) and behaves stoichiometrically as if it is part of a multiprotein complex [599]. A milder antagonist of Arm is dCBP (cf. Hh and Dpp pathways): dCBP binds Pan and acetylates a lysine in Pan’s Armbinding domain, thus decreasing Pan’s afﬁnity for Arm [4533]. Still another means of inﬂuencing Pan in mammals and nematodes (and maybe ﬂies) is by phosphorylation of Pan, which blocks Wg signaling [1996]. The ability of

295

Pan to bind Arm may also be inhibited by a small (81a.a.) Arm-binding protein “Icat” (Inhibitor of β-catenin and TCF-4) found in Xenopus [4232]. In ﬂy embryos, the zinc-ﬁnger protein Teashirt is an auxiliary Arm-binding partner and modulator of Wg signaling [1344, 1345], and the nuclear protein Lines may be playing a similar role [1759]. Pan can also regulate target genes negatively (in response to Wg signaling) by competing with activators at overlapping binding sites in their promoters [3386] – the phenomenon of “quenching” [1600].

3.L. Naked cuticle (Nkd, named for embryo defect) is a 928-a.a. hydrophilic protein with a putative Ca2+ binding (EF hand) motif, implying ion ﬂuxes in Wg signaling [4863]. The nkd gene is activated wherever Wg signal is received, but nkd GOF transgenes cause wg LOF phenotypes. Thus, Nkd seems to be part of a negative feedback loop (cf. Slimb [4038]) – analogous, perhaps, to Dad’s role in the Dpp pathway. However, disabling nkd in somatic clones has little effect, so it may act redundantly with another gene [4863]. Nkd binds Dsh and appears to act at the level of Dsh (or immediately downstream) in the Wg pathway [3659]. 3.M. Target genes include the following, plus possibly dE2F (turned off at the wing margin) [1126, 2079, 3979]. wg itself in a paracrine [3690] but not autocrine manner at the wing margin. This link apparently does not operate within the blade proper [353]. In the notal part of the wing disc, Wg wg ; however (for some unknown reason), this feedback loop only pushes the wg-on domain a few cells beyond its pannier-dependent realm [4369], instead of expanding to ﬁll the entire disc. Wg Dfz2 (partially) in wing discs [595]. Wg Dfz3 in wing, leg, and eye discs [3977]. Wg arr in leg discs [4570], which suggests that wgon cells should be relatively deaf to their own signal. Wg nkd wherever Wg is received [4863]. Wg dpp in leg discs [487, 2059, 2082, 2954, 4277] and eye discs [4387] except at the anterior margin [1780]. In contrast, Wg dpp in the optic lobe of the brain [2130]. Wg aristaless in leg and wing discs (at a certain threshold) IF Dpp is also present [643] . Wg dachshund in leg discs (at a certain threshold) IF Dpp is also present [2456]. Wg Distal-less in leg discs (at a certain threshold) IF Dpp is also present [2456] , and the same is true for antennal anlagen [1037]. In wing discs, however, Wg Distal-less independently of Dpp [595] .

1. Wg [595]

2. 3. 4. 5. 6.

7. 8. 9.

296

10. Wg hairy in leg discs IF Hh is also present [1778]. 11. Wg H15 in leg discs [3762] . 12. Wg omb in wing discs [1626], although the control appears to be indirect [3978]. 13. Wg {achaete and scute} at the wing margin [3691] and prospective notum [3373] , but Wg {achaete and scute} in the developing eye [597]. 14. Wg {araucan and caupolican} at the wing margin [1537]. 15. Wg string (an indirect target that is turned off by achaete and scute) at the wing margin [2079, 3979]. 16. Wg Delta at the wing margin [595] . 17. Wg Serrate at the wing margin [595] . 18. Wg vestigial in the wing pouch [734] . The same link can be artiﬁcially triggered in leg discs [2753], where it requires the cooperation of Dpp [2754] . 19. Wg scalloped in the wing pouch [353] . 20. Wg homothorax in the wing pouch [157] via vestigial (at least in part), but Wg homothorax in the wing hinge [678] and head capsule [3380]. 21. Wg ventral veinless (a.k.a. drifter) in the wing pouch [703, 990] . 22. Wg vein in the wing pouch [4604] and dorsal head [80].

APPENDIX SIX. SIGNAL TRANSDUCTION PATHWAYS

apterous in the wing pouch in 2nd instar but 23. Wg not thereafter [4683]. 24. Wg spalt in the notal portion of the wing disc [987]. 25. Wg BarH1 in the notal portion of the wing disc [3763]. 26. Wg orthodenticle in the dorsal head [3665]. 27. Wg mirror in the developing eye [1781] . 28. Wg WR122-lacZ (an enhancer trap expressed at the equator) in the developing eye [1781]. 29. Wg eyeless in eye discs IF Dpp is also present [2465]. 30. Wg atonal in the developing antenna [2055].

3.N. Summary of the basic chain: Wg Dfz2 Arr Dsh Sgg Arm Pan {target genes}. Binding of Wg to Dfz2/Arr alters Dsh so that it can disrupt the complex of Sgg, dAxin, E-APC, PP2A, Slimb, Arm, and ubiquitinating enzymes. Dsh causes an unknown kinase to phosphorylate Sgg, thus lowering Sgg’s own kinase activity. PP2A removes the phosphate that Sgg had put on a protein (dAxin? E-APC? Arm?) that keeps Arm in the complex. Arm is freed and escapes proteolysis. Its concentration rises. Arm displaces Gro from GroPan dimers, and the Arm-Pan dimers activate Wg-target genes [269]. How Wg represses target genes is unclear [1759].

APPENDIX SEVEN

Commentaries on the Pithier Figures

The following musings concern certain ﬁgures that warrant further scrutiny. Through these distillations, I attempt to draw some general conclusions about how the ﬂy’s control system operates and why it evolved this way. Some of the annotations also offer historical perspective.

Figure 2.1 One corollary issue (symbolized by the hourglass) is: how do cells measure time over periods longer than a mitotic cycle? Cells presumably need to do so in order to know when to stop dividing and start differentiating. Possible timekeeping devices include the “POU Hourglass,” which limits the number of mitoses in certain neuroblasts in the ﬂy CNS [313, 314, 3713, 4804, 4816]. This clock gauges the declining amount of the POU-domain proteins Pdm-1 and Pdm-2. Mammalian oligodendrocytes use an “HLH Hourglass” that triggers differentiation when the amount of speciﬁc HLH-domain proteins drops below a critical threshold [2293, 2294]. An oscillator based on this sort of mechanism may be involved in vertebrate somitogenesis [945]. Other protein clocks appear to count mitoses and meioses leading to sperm and egg differentiation [1762, 4631]. Another strategy involves using a cascade of transcription factors to trigger different events in different phases of the cascade [485]. For RNA clocks, see [223, 1206], and for general discourses on (noncircadian) timekeeping, see [867, 1227, 2762, 3767, 4011]. A deeper issue concerns how structures are represented abstractly in the genome [462, 2724]. Given that we know most of the genes involved in bristle development (cf. App. 3), we can begin to ask how “bristle” is “written” in “gene language.” The answer is not obvious. Clearly,

a bristle is the outcome of a chain of cellular events, but each event uses genes that are also used elsewhere. No “master gene” exists here, nor is there a hierarchy of dedicated subordinate genes [1114, 2410]. If we could interrogate the shaft cell, for example, it would be unable to deﬁne its identity without reciting its history. In this sense, the genes act as a program [98], not as a blueprint [136, 1094]. How the “Make a bristle!” program is encoded remains enigmatic.

Figure 2.2 The Nuclear Notch Model is considered proven [443, 711, 1329, 2514], although Su(H) can affect transcription without Notch [1329, 2129, 2255, 2540, 2941]. Catalytic activation of Su(H) by Notch (or a binding partner) is still possible but hard to reconcile with the pathway’s sensitivity to the dosage of Notch and Delta [118, 1797]. The ability of Notch’s intracellular domain to act alone quashed the idea [1204, 1742, 1899, 1900] that Notch’s primary role is in cell adhesion [112, 3022, 4161]. Nevertheless, Notch is involved in epithelial-mesenchymal transitions [1737, 1744, 4269], and its removal may foster neuroblast delamination [4129]. Another old idea was that Notch’s role is to keep cells in an undifferentiated state [112, 113, 1308]. That idea is contradicted by the ability of Notch-activated cells to differentiate as socket or sheath (cf. Fig. 2.1). This ﬁgure was adapted from [1251] and op. cit. Figure 2.3 In other signaling pathways, the inputoutput complexity is distributed among many proteins (cf. Figs. 5.6 and 6.12), not concentrated in one big molecule as here (although the cytoplasmic domain of Egfr is also intricate [2007, 3184]). The variety of domains suggests that Notch was cobbled together evolutionarily from parts of other proteins [3289], and the multiplicity 297

298

APPENDIX SEVEN. COMMENTARIES ON THE PITHIER FIGURES

of each type of domain implies repeated episodes of tandem duplication [1824]. Our grasp of this gadgetry is hampered by our ignorance of “cellomics” [1389] – the 3-D protein shapes of the cogs and their gearing stoichiometry [58, 136, 1249]. See [75, 459, 3271, 4206, 4207] for Notch’s nuances and [1758, 2310, 4041, 4499, 4837, 4838] for the larger issue of input-output kinetics. John Dexter, a student of Morgan’s [2283], found the ﬁrst Notch mutant in 1913, and Morgan himself discovered an allele (later lost) in 1915 [470].

Figure 2.4 Despite knowing the DNA sequence and binding sites for the promoter in g, we remain ignorant about how it works [871]. Indeed, we are pretty clueless about “promoter logic” in general [393, 2836, 4165, 4755]. Yes, we know that trans-acting factors along the DNA compute a certain output of mRNA [1230, 4464, 4836], but we still need to ﬁgure out how they interact physically with one another and with the polymerase machinery (cf. Ch. 8). The gene complexes in a and b probably evolved from single genes that duplicated repeatedly [1877, 3075, 3178, 3616, 3982, 4617], as apparently happened in the Hox complexes [146]. Unequal crossing over was ﬁrst deduced at the Bar locus by A. H. Sturtevant in 1925 [4179], and tandem duplications were conjectured for the Bithorax Complex by E. B. Lewis in 1951 [2505]. Evolutionarily, such events may have been facilitated by transposons [1401, 1416, 1602]. After a duplication, the paralogs would act redundantly [1517, 1824, 3384, 4584] until cis-regulatory regions have time to diverge [1114, 1419, 2630, 4260, 4519]. Divergence could occur by (1) asymmetric loss of old cis-control sites [694, 1265, 2316, 2629, 4266] or (2) asymmetric creation of new ones that capture foreign trans-acting regulators [1114, 1151, 1347, 3379, 4259, 4704]. Point mutations could lead to new sites that are small (≤∼10 b.p.) [662, 1477, 2597, 2616, 2763, 3477], but larger sites probably arose via cut-and-paste transpositions [1046, 2231, 2236, 2480, 3075, 3816, 3840] or inversions [593, 666, 2583, 3615]. To the extent that nearby genes share cisenhancers, each complex will tend to remain intact [699, 871, 1112, 1579, 2508, 2672]. Remaining mysteries that are worth pondering include 1.

How do tandem duplicates avoid cosuppression [339, 340, 1823, 1825, 3231, 3396]?

2. 3.

Why did colinearity evolve in Hox complexes but not elsewhere [1113, 2508, 2672, 3494]? How on earth did groucho get into a complex of unrelated (bHLH) genes whose products appear to mainly interact with Groucho at a protein ( vs. cisgenetic) level?

See [146, 2538, 4786] for other case studies, [2034] for the idea of “gene sharing,” and [662, 866, 968, 1440, 3144, 3496, 4298] for overview.

Figure 3.1 No sketch or photo can do justice to the splendor of this ﬂy. In 1683 (the earliest known account of this species), Christian Mentzel (1622–1701) waxed poetic [3361]: Its eyes are purple. The back is yellow and gibbous. The tail also is yellow, signed by six dark wasp-like stripes or lines. The small wings are a little longer than the body and very transparent. In bright daylight they are splendidly iridescent like a rainbow. . . . Back and head are full of certain bristles on both sides.

In 1764, W. F. von Gleichen offered another ode [2982]: The head, which is somewhat broader than the thorax, is shaped towards the rear like that of other flies, but by far more beautiful in color and the red eyes on both sides of it are very ornamental. The latter are separated by a broad band which is set with fine hairs on both sides. . . . The brown chest-plate looks like a tile, arched and covered all over with small hairs, especially towards the narrower end. The six long hairy legs are attached to the thorax. . . . Two small bubblelike balancers can be seen between the middle and rear thigh and on both sides under the chest-plate. . . . The body is very hairy and divided into five large rings. It reflects through the two long net-like and iridescent wings which are interwoven by many nerves.

The ultimate paean, however, is Curt Stern’s 1954 classic,“Two or three bristles” [4096]: I marvel at the clear-cut form of the head with giant red eyes, the antennae, and elaborate mouth parts, at the arch of the sturdy thorax bearing a pair of beautifully iridescent, transparent wings and three pairs of legs, at the design of the simple abdomen composed of a series of ringlike segments. A shining, waxed armor of chitin entirely covers the body of the insect. In some regions this armor is bare, but in other regions there arise short or long outgrowths -- the bristles -- strong and wide at the base and gently tapering off to a fine point. Narrow grooves, as in fluted columns with a slightly baroque twist, extend along their lengths. A short stalk fits each bristle into a round socket within the body armor so that the bristle can be moved within this articulation. There is a regular arrangement of these bristles. . . . With surrealistic clarity the dark colored bristles and hairs project from the light brownish surface of the animal, delicate but stiff, in rigid symmetry.

In fact, bristle grooves do not twist helically around the shaft, although it is easy to see how Stern could have thought so based on side views. Rather, they converge like chevrons at a seam along the top [4639, 4641]. Newcomers may wonder: Why did T. H. Morgan choose this inconspicuous species as his experimental

APPENDIX SEVEN. COMMENTARIES ON THE PITHIER FIGURES

organism? In his 1925 opus with coauthors Calvin Bridges and Alfred Sturtevant, Morgan explains [2951]: The rapid rate of multiplication of the vinegar fly, owing to the large number of offspring produced, and to the brevity of the life cycle, makes it excellent material for genetic purposes. At room temperature or a little above (24 to 25◦ C), it takes only about 10 days to pass through a complete generation from imago to imago. Under favorable conditions twenty-five generations may be bred in a year.

Figure 3.4 The AS-C’s ∼8 cis-enhancer elements (a) are using an “or” logic: if any of them is activated by a trans-acting factor (cf. Fig. 6.14), then the cell will become proneural via expression of ac and/or sc. It is easy to see, therefore, how evolution could insert or delete such modules without harming the function of the locus [1094] – a system property called “ﬂexible robustness” [1440]. Indeed, this complex is one of the clearest examples of cis-regulatory modularity in any metazoan [968]. Figure 3.6 The feedback circuit in a blurs the distinction between “competence” (mediated by the AS-C) and “determination” (mediated by the Delta-Notch duality) [1455, 1654]. Its bistable quality ensures discrete cell types [2162, 2163], but the ﬁnal pattern of those states is somewhat stochastic. Hence, ﬂy genes do not dictate bristle sites any more than human genes dictate the swirls of ridges on our ﬁngertips [923, 1159, 1606]. This notion of “emergent properties” in control systems was a favorite theme for the great embryologist Paul Weiss [4593, 4594]. Figure 3.10 These patterns are obviously much richer than the emblematic French ﬂag of Wolpert’s Positional Information Hypothesis [4723], and it is likely that a variety of tricks (aside from gradients) help organize the bristles (cf. Fig. 3.11) [1805]. For example, SOPs might align into transverse rows via ﬁlopodia [3051] or via homophilic binding proteins on the sides of each cell [3048]. Similar strategies may be used in the pedicel of the haltere, where sensilla campaniformia align into strikingly similar transverse rows [1866, 3623]. Figure 3.12 The importance of temporal factors in gene expression (especially “rate genes”; cf. i) was a favorite theme for the controversial geneticist Richard Goldschmidt (1878–1958) [1521, 1524, 3398]. His mental playground was “physiological genetics” [1520]. Upon that foundation the ediﬁce of “developmental genetics” was built [3494] by Curt Stern (his prot´eg´e) [4095, 4100, 4104], Ernst Hadorn [1667], C. H. Waddington [4510, 4513, 4515], and others outside the Fly World [2309, 2692]. Indeed, “heterochronic” mutations that tinker with the timing of events are thought to have been instrumental in evolution [978, 1114,

299

1581, 2788, 2789]. Two intriguing mutations of this kind have been documented in butterﬂy wings, where a time window appears to govern scale pigmentation [2278].

Figure 4.1 Histoblast nests are replaced by ectopic discs when the Bithorax Complex is disabled [828, 1285, 2670, 2745, 3930, 4434] (cf. Fig. 4.2), a default state that has intriguing evolutionary implications [263, 664, 682, 1429, 2503, 4550, 4552]. For example, Ubx bxd LOF adults have four pairs of legs [2506], a condition that predates the order Insecta [4266]. N.B.: In wild-type embryos, the leg discs are only partly within the neuroectoderm [389] (not totally immersed as depicted here). Figure 4.3 Despite its demise in 1969 and its formal “burial” in 1978 [4346], Stern’s Prepattern Hypothesis was resurrected in the 1980s as a way of explaining embryonic segmentation (see text), where odd sets of signals along the anterior-posterior axis of ﬂy embryos demarcate body segments. It lives on today in derivative models that explain bristle pattern formation in terms of upstream “prepattern” genes (cf. Fig. 6.14c and Table 6.2). Indeed, both models have proven useful in different contexts (cf. Fig. 8.3). Quirks of antenna-leg topology (a) include (1) unlike walking legs, antennal “legs” face up as shown [2933, 3441, 3775, 4145, 4212] because eye discs rotate ∼180◦ after arising, thereby inverting their dorsal-ventral axis [4146]; (2) in more extreme genotypes, the sternopleura (a sclerite proximal to Co) replaces anterior head vibrissae [1461]; and (3) joint membranes that arise at the arista base [1260] resemble tarsal joints but may differ developmentally. Given the homology between the arista and the claws [620, 1085], it seems odd that the A/P lineage boundary bisects the claws [2443, 2449, 4076] but skirts the arista entirely [2931, 4146, 4148]. Nevertheless, tarsal and aristal primordia are similarly bisected by the engrailed on/off line [1697]. The arista may be using a recent gene circuit that evolution inserted into the tarsal algorithm [1085, 1119]. Antenna-leg homologies are further explored in Chapter 8. Figure 5.3 Both models assume that cells adopt fates due to “social” signals that, ultimately, are properties of the whole population (cf. Ch. 1). One beneﬁt of this strategy is robustness [1839]: no cell is ever given so much authority that its malfunction is catastrophic [858, 1440]. Thus, the system withstands sporadic cell death [1776, 3440, 3449], deviant mitotic rates [2935, 3947--3949, 3961, 3962, 3965], and even massive tissue loss [525, 1299, 2698]. For the same reason, discs have tolerated allometric changes during evolution [4114]. This tenacity suggests that the system

300

APPENDIX SEVEN. COMMENTARIES ON THE PITHIER FIGURES

is goal-driven [2237] (cf. Driesch’s entelechy [2989, 3743]), but in fact it must be rule-driven [1558, 2160, 4605] because the abnormal limbs that it produces when perturbed (c–e, g–i) obey Bateson’s Rule [240, 539]. Generative rules of this kind constrain the paths that evolution can take [56, 622, 1583, 1584, 1690, 2767] – an insight that was famously formalized in Waddington’s “epigenetic landscape” [1114, 1488, 4513].

Figure 5.6 Despite their differences, these pathways have essentially the same input-output capability (cf. Figs. 2.2 and 6.12) [3287, 3288], so the evolutionary decision to use one or another in speciﬁc patterning events was probably arbitrary and accidental [2301]. The ability of so many proteins to bind one another makes these networks highly nonlinear [2220]. The transient aggregates of transducer proteins have been called “transducisomes” [556, 4405, 4406] in analogy with conglomerate machines like the ribosome [687], replisome [187, 2490], spliceosome [563, 3996, 4061], enhanceosome [649, 1317, 2109, 2826], and transcriptosome [1703, 2304, 2488, 3279], not to mention the mammoth Mediator [412, 2665] and the zoo of chromatin-remodeling contraptions [1, 2227, 2228] (e.g., Brahma [849, 908, 2114], MSL [1633, 2183, 3760], NuRD [41, 2271], Pc-G [2622, 3869], SWI/SNF [252, 1752, 4199], and even one that is speciﬁc to the ﬂy’s 4th chromosome [2412]). In general, the beneﬁts of multiprotein consolidation include (1) greater speed by conﬁning reagents [1106, 3651]; (2) greater efﬁciency by managing the order, orientation, and stoichiometry of reactions [1062, 3297, 3299, 3834, 4406]; (3) greater speciﬁcity by increasing the requisite number of docking domains [3874, 4252, 4253]; (4) reduced interpathway cross-talk by forming insulated corridors [1062, 1389, 2007, 3297, 4896]; and (5) sharper output by fostering allosteric cooperativity among the partners [829, 1555, 3473, 4597]. Hence, the old view of the cell as a “soup” of freely diffusing molecules [565, 1146, 2875, 3302, 4064] is giving way to the idea that cells rely more on “solid-state” chemistry [36, 58, 441, 442] and jigsaw-puzzle computations [440], although the interactions may still be fairly sloppy [2236]. Another design feature of these (and other) pathways is a reliance on phosphorylation. Adding or removing phosphates is an easy way of (1) altering docking domains [2136, 3874, 4200], (2) amplifying signals [441, 1950, 2311], (3) creating feedback loops [1292, 1675, 2943, 3010, 3347], (4) recycling transducers [59, 4606], and (5) modulating transcription factors [3874, 4176]. Moreover, proteins that have multiple phosphorylation sites can integrate the various positive and negative inputs combinatorially [440, 3184] in

the same way that a neuron “computes” an output from its multiple inputs [2275, 2276].

Figure 5.11 Despite recovering so many genes with quasisegmental expression patterns, we still do not know what makes one leg segment different from another. All leg segments are thought to use the same kind of gradient to specify cell positions along their length [386, 2425, 4143], but if that is true, then different segments should need different identity codes so that they can interpret the iterated gradient in different ways. In fact, though, few homeotic mutations have been found that transform one type of leg segment into another (see text). We also do not know what genes create the membranes between segments [3421]. For inventories of segmentation defects, see [1810, 4512, 4515]. For evolutionary context, see [10, 4230]. Similar annuli arise in the legs of grasshoppers [718, 3971] and cockroaches [3132] but in an odd sequence [234]. The tarsus of ancestral arthropods was unsegmented [415], and it may have subdivided by heterotopic deployment of spineless from the antenna to the leg (cf. Ch. 8) [1119]. Mapping of unassigned rings (from photos) is based on layouts in [834] (his Fig. 11d), [1311] (their Fig. 14d), [2287], and [4484]. Circuitry links are mainly from [344, 1573, 2287, 3523, bab” and “al bab” links, which help 3525]. The “dac delimit the bab-on domain, are from J. P. Couso (pers. comm.), who also has found that al’s inﬂuence on bab from a distance is mediated by Egfr. The question mark (lower left) concerns how Ser and Dl act. GOF studies indicate that either Ser or Dl alone is sufﬁcient to induce expression of downstream genes [3525], implying “or” logic. However, each gene has a LOF phenotype (absence of joints), ruling out a simple redundancy and implying “and” logic [344]. Although fringe is crucial for joint formation [344, 3525], it is excluded here because its expression data contradict its LOF defects [999, 1988, 3525]. Also omitted: 18 wheeler and klumpfuss (LOF alleles suppress joints but rings are unmapped) [344] , Arrowhead (spiral stripe vs. ring) [928], BarH2 (same as BarH1) [2287], enabled (≈ dpn but not on in claws) [3505], extradenticle (Exd is nuclear in hthon domain and cytoplasmic elsewhere) [9] , grain (Fe and Ti spots, T5 ring) [509], l(3)1215 (probable wide-zone type) [539, 792, 1810], mβ (≈ m8 but stronger and detectable proximally) [871] , orthodenticle (very proximal crescent) [4654], pipsqueak (≈ Dll ) [4567], prickle (uniform but absent from tarsal joints) [1641], Toll-like receptor (≈ HZ76) [760], Iro-C enhancer-trap lines iroSc2 and iroB6.8 (≈ ss) [3079], and enhancer-trap lines c803 (≈ Dll ) [2684], 05271,

APPENDIX SEVEN. COMMENTARIES ON THE PITHIER FIGURES

07022, and k10209 (≈ ap) [1397]. See FlyView ( ﬂyview.unimuenster.de) for more enhancer-trap expression patterns [2027] and [4048] for enzyme patterns. A similar diagram of some of these domains is given in [1341, 3523] with further annotations.

Figure 6.2 The kaleidoscope of patterns collected here reveals how balkanized the disc is, despite the cellular homogeneity seen in ordinary histological preparations [981]. Until the 1980s, such differences could only be inferred from indirect approaches such as fate mapping. Now we know that cell fates reﬂect identities that are cobbled together from subsets of genes. In short, circuitry dictates destiny. The mythical “prepattern” prophesied by Stern [4095] turned out to be a rich “landscape of transcriptional regulators” [981] (cf. Fig. 6.14). Genes omitted (relation to depicted patterns is in parentheses where “not-” means “complementary to”): extradenticle (Exd-nuclear ≈ not-Dll but patchy in notum) [130] , four jointed (≈ vg) [483] , invected (= en) [842], master of thick veins (≈ not-tkv in the pouch) [1327], mβ (≈ emc but also on in P interveins) [871], ptc (≈ dpp but weakly on in A region; cf. Fig. 6.3) [4188], scalloped (= vg) [1686] , scute (= ac [3689] , Serrate (= ap in 2nd and early-3rd instar) [163] , spalt-related (= spalt) [984], Tolllike receptor (hinge, part of wing margin, and 2 large notal spots) [760]. Aldehyde oxidase and other enzymes (not shown) have unique patterns [2342, 4049]. Nubbin is expressed and required in the hinge as well as the pouch (not shown) [157, 3103], notwithstanding reports to the contrary [793] – a fact that has caused some confusion in circuit analysis (compare [2252] vs. [3104]). For galleries of expression pattern images, see [353, 834, 2092, 2252, 3089, 4683], and for enhancer-trap patterns, see [4210] and FlyView ( ﬂyview.uni-muenster.de) [2027]. The link “Knot bs” [4479] was omitted due to space limitations, and bs is also regulated independently by Dpp [3145]. Figure 6.3 These gradients bear a striking resemblance to Wolpert’s scheme for positional information (cf. Fig. 4.3b). However, there is no evidence that (1) cells “record” any morphogen levels as lasting memories or that (2) cells “interpret” any levels aside from the few that delimit target gene expression zones [224, 3087]. Some of the A-P zones are later subdivided to create wing veins (cf. Fig. 6.11), but even during that “read out” phase it is not clear that any cell actually knows where it is. Rather, cells seem to adopt progressively more restricted qualitative states. Such states surely depend on position, but not on PI per se [2448]. In general, the patterning mechanisms discovered in discs show features

301

of both PI (the gradient stage) and prepatterns (local cues ≈ “singularities”). As for why the A-P axis needs two gradients, one idea is that cells cannot discriminate Dpp levels near the source and hence must rely on the higher-resolution Hh gradient [224, 865]. However, the same argument could be made for the D-V axis, which manages very nicely with only one gradient. To the extent that these morphogens dictate cell fates over large distances, their source zones are acting as “organizers” [1040, 1699, 3745, 4510]. In 1936, the renowned scholar Joseph Needham reasoned that organizers probably use sterols as morphogens [3063], thus anticipating the 1996 discovery of Hh’s cholesterol adduct [3434]. While Wg controls vg in most of the pouch [2254] (notwithstanding suspicions to the contrary [835]), vg is activated by Notch at the D/V boundary [2216, 3091, 3490, 4849] via a separate cis-enhancer [2219, 2254, 3089, 4684]. Cells that receive both Wg and Vg adopt a “blade” fate, whereas those that get only Wg (from a wg-on ring not shown) adopt a “hinge” fate [2252, 2570] via homothorax [678] and respond by proliferating [3088]. Details omitted: (1) A vs. P halves of the pouch differ in size and shape [3972], (2) A vs. P Dpp gradients differ in shape and slope [1327] , (3) all gradients are probably exponential (e.g., see [1169, 2832, 4251, 4265]), and (4) the dpp-on stripe “varies in width from ∼2 cells at the center of the wing pouch to ∼8 cells at the periphery” and even vanishes at one point where it crosses the wing margin [3497] (but see [4251]). This sector shape recalls leg disc’s wedges (cf. Fig. 5.8 and 2nd-instar wg-on sector in Fig. 6.2) and suggests a common geometry for leg and wing patterning. Registration of the dpp-on zone with vein 3 is based on ref. [350], but in other specimens this zone seems to ﬁll the entire area between veins 3 and 4 [4188]. N.B.: The “Hh dpp” and “Dpp omb” links work similarly in wing and leg discs (cf. Fig. 5.8), but the leg’s “Hh wg” link is inoperative in the wing. Other wiring differences include (1) vg is not turned on in leg discs [4681], (2) spalt is only on in a few proximal cells there [220], and (3) Dll is solely regulated by Wg in the wing [3089] but is under dual control ({Wg and Dpp} Dll ) in the leg (cf. Ch. 5) [1037]. The “En dpp” link may not actually function anterior to the A/P line as shown here. Seth Blair (pers. comm.) does ﬁnd evidence for such repression when he monitors dpp expression with lacZ driven by the lone dpp enhancer “BS3.0” (the usual method [347]), but not with more reliable inserts of lacZ at the dpp locus (i.e., enhancer traps). If the anterior

302

APPENDIX SEVEN. COMMENTARIES ON THE PITHIER FIGURES

“En dpp” effect is spurious, then the Registration Riddle becomes moot (see text).

Figure 6.7 The creation of a third type of cellular state (here a fringe of bristles) at the interface between two others is a common theme in development [825, 3119, 4075, 4114, 4397] (e.g., the vertebrate neural crest [3853, 4135, 4868]; cf. Figs. 5.12c, 6.6b, and 6.11). This “Stratiﬁcation Device” is the epitome of an emergent property [2989, 4098]. It was used as a bootstrapping trick in Meinhardt’s Boundary Model II in 1983 (cf. Fig. 5.4) [2808], and its power as a tool for tissue elaboration was recognized much earlier by Paul Weiss in 1961 [4592] and by Curt Stern in 1936 [4088]: “. . . any physiological activity of a cell or a cell area, if liable to be inﬂuenced by the neighboring areas, will lead to new differences. The part of a homogeneous area A bordering on another area B will become different from that part of A which borders on C.” A vivid incarnation of this phenomenon was documented by Whiting in 1934: a novel zone of pigmentation arises in genetically mosaic wasp eyes when pigment precursor molecules diffuse across the border between territories [4634]. N.B.: Partial elimination of the margin in Lyra mutants [2, 4] turns out to be due to a neomorphic mutation at the senseless locus [3128], which normally has nothing to do with margin formation [3127]. Figure 6.9 These dualities (a.i and b.i) underscore the progressive nature of disc development (cf. Fig. 5.5 for the leg disc). After establishing two cardinal regions (wing vs. notum, or eye vs. antenna), each disc is further subdivided into parts (cf. Figs. 5.8, 6.3, 6.14, 7.3, and 8.3) that are then patterned at a ﬁner scale (cf. Figs. 3.3, 3.8, 3.9, 6.11, 7.7, and 7.9). The notion that embryos are built stepwise by this kind of elaboration and embellishment [825] was asserted by Hans Driesch in 1894 [1101] (as translated by T. H. Morgan [2945]; cf. a different translation by Curt Stern [4096]): Development proceeds from a few prearranged conditions, that are given in the structure of the egg, and these conditions, by reacting on each other, produce new conditions, and these may in turn react on the first ones, etc. With every effect there is at the same time a new cause, and the possibility of a new specific action, i.e. the development of a specific receiving station for stimuli. In this way there develops from the simple conditions existing in the egg the complicated form of the embryo.

N.B.: The Iroquois Complex (not shown) acts downstream of Vn (Vn Iro-C notum state) [4543]: ara GOF transforms wing to notum [4543], and Iro-C LOF clones in

the notum switch cells to “hinge” fate [1060]. Transdetermination frequencies can deviate considerably from the trends depicted here, depending on genotype, culture conditions, and other factors [3883].

Figure 6.10 Other examples of this Venn logic include (1) wingless in the notum, which is turned off wherever Pannier and U-shaped overlap (cf. Fig. 6.14) [1380, 1671, 3764]; (2) spalt in the antenna, which is only turned on where Homothorax and Distal-less overlap (cf. Figs. 7.3 and 8.3) [1085]; and (3) Extradenticle, which appears throughout the leg and antenna but only goes to the nucleus where Homothorax is also present [8, 9, 130, 677, 2673, 3589]. In the Vg/Sd case, a third state emerges at the overlap between two cardinal states. Evolution has undoubtedly “invented” a wide spectrum of genetic circuits by playing with this sort of genetic “Kaleidoscope Toy” (see [2353]). The ultimate example may be the eye, where ≥4 “early eye” genes must be coexpressed in order to specify eye identity [2353]. A variation on this theme is the use of “not” conditions. For example, Hairy stripes suppress achaete on the tarsus (Hairy achaete) to leave a complementary pattern of Achaete stripes (cf. Fig. 3.9), and Extramacrochaetae performs a similar masking function on the notum (cf. Ch. 3). Like feedback circuits (cf. Fig. 3.6), combinatorial control mechanisms defy reductionism because, in this case, the whole is literally more than the sum of the parts [2407, 4644]. When two genes can only function in combination (as here), each can be turned on separately in other places without harming development [694, 1046]. This freedom helps explain why so many genes are expressed in cells where they have no apparent function [1253, 1654, 2845] (e.g., period [1693, 1694], sevenless [194, 4363], and string [2480]). To wit, evolution is evidently “tinkering” harmlessly [1114, 2005, 3494], and some of these playful links may lead to opportunistic pleiotropy [815, 1046, 1865, 2845, 4110]. Examples of such tinkering are apparent in many “housekeeping” enzymes (e.g., aldehyde oxidase [2339, 2340, 4050, 4051], glucose-6-phosphate dehydrogenase [924], isocitrate dehydrogenase [2341], NADP+ -dependent malic enzyme [1235, 2344], and 6-phosphogluconate dehydrogenase [924]). The functional irrelevance of these patterns is shown by their liberal evolution in the leg, wing, and eye discs of various ﬂy species [4048, 4049]. Similar frivolity is familiar in the pigment patterns of dogs, cats, and other mammals [3011--3013, 4819]. How Sd interferes with gene expression is unclear [3936]. One possibility (shown here) is that Sd recruits a co-repressor (“?”). This notion ﬁnds support in (1)

APPENDIX SEVEN. COMMENTARIES ON THE PITHIER FIGURES

the ability of excess Sd to turn off target genes even when Vg is present, which suggests a Sd partner aside from Vg [1686, 4463]; (2) the identiﬁcation of a candidate corepressor for Sd’s human ortholog TEF-1 [727, 728]; and (3) the existence of a dominant vg allele (vg79d5 ) whose Vg protein binds Sd but may lack an activation domain [3291], thus mimicking a co-repressor. This ﬁgure was adapted from [445, 1686]. N.B.: The exclusive need for vg in the wing is shown by vg null ﬂies, which lack wings and halteres but otherwise look surprisingly normal [3291]. The Vg circle in c should more aptly be a subset of the Sd circle (not an overlapping set) because there is no known region where vg is on without sd also being on [3936], although one may yet be found. Vg’s subcellular location sans Sd (d) was assessed in cell culture [1686] and with a Vg construct lacking its Sd-interaction domain [3936]. It is not known whether Sd uses Vg-like partners in regions where it is on but vg is off. Vg alters Sd’s DNA-binding speciﬁcity [445, 3936], and Vg:Sd stoichiometry is critical for all known functions of both genes [2570]. The above reasoning has been tested by making a chimeric Vg-Sd construct [4054]. Expression of that fusion protein in various tissues successfully mimics the effects of Vg/Sd heterodimers.

Figure 6.11 The ﬂow of this circuit (from broad to narrow districts) resembles the embryo’s segmentation hierarchy (cf. Fig. 4.2) [981, 984, 4188], and it nicely shows how deep a regulatory network can be [968]. It is surprising that evolution opted for such complexity to allocate only two cell states, given that the same periodicity could theoretically have been created much more simply [1805, 2806]. Genes omitted: abrupt (∆L5) [320] , caupolican (≈ ara in its expression and regulation) [984] , emc (≈ vein at Ev, but ≈ mβ at 30 h AP) [205], nemo (≈ argos at 30 h AP) [4475], scute (≈ ac) [320], short gastrulation (≈ tkv) [4831], spitz (≈ Star) [1643], ventral veinless (≈ Dl at 24–28 h AP but off along margin) [984] . It is unclear whether Dl is actually expressed in ∼3-cell-wide “vein” stripes as probed by anti-Dl antibodies [205] or in ∼8-cell-wide provein stripes as probed with antisense RNA [989]. The “kek1 Egfr” link is inferred from (1) Egfr downregulation where kek1 is expressed, (2) kek1 LOF effects elsewhere [1445], and (3) kek1 GOF effects on veins [4604]. The lack of a kek1 LOF vein phenotype may be due to redundant paralogs [1445]. The antagonism between rho and net [466] is shown in c but omitted from e. Evidence exists for a “net bs” link [320], which could be indirect (i.e., “net rho bs”). For further interactions, see [980, 995,

303

1041, 1368, 4189]. Regulation of caup appears identical to ara,

and both genes require high levels of Dpp (not shown) via a route that somehow bypasses omb and spalt [1537]. Aside from ptc LOF (cf. Fig. 6.6e, f), other A-region clones that induce L3 outside their perimeter (not shown) include DC0 null [3238], DC0 null dpp null (but not singly dpp null ) [2058], and en GOF [4848]. An ectopic L2 is induced at the front edge of Distal-less null clones that reside anterior to L2 [1561]. Egfr LOF clones can elicit an ectopic vein outside their perimeter in the vicinity of any extant vein when they overlap it [1042], and knot LOF clones (marked here as “i”) can extend their ectopic veins a few cell diameters into surrounding wild-type territory [2894].

Figure 6.12 More RTK molecules can be deployed to improve sensitivity at minimal cost in energy because subsequent steps are only executed if receptors dimerize. This “assembly on demand” strategy is a clever engineering trick [441]. Another trick is the use of a 2-D surface (the membrane) to increase the likelihood of productive collisions among recruited components during the assembly process [441]. The intricacy of this network recalls Dearden and Akam’s lament that such diagrams resemble “explosions in a spaghetti factory” [1010]. Their remedy is to go “in silico” because computers can pick up where our intuitions leave off [1758, 4499]. Time will tell whether the cell’s full “instruction manual” is so sophisticated that only math wizards can hope to understand it. For now at least, any literate car mechanic or house electrician should be able to ﬁgure out the basics. Why is this pathway so complex when its inputoutput relationship seems to be a trivial relay (devoid of true computation) like a one-step JAK-STAT [958] or Notch [446] pathway (cf. Fig. 2.2) [1846]? Proposed answers include (1) the multitude of nodes allows for interpathway cross-talk [215, 375, 1663, 2892, 3622], (2) the negative loops offer ﬂexibility for operating in either digital (solenoid) or analog (rheostat) mode [1222, 1292, 2654], and (3) the added “frills” are useful for modulating the duration and amplitude of the signal [2701, 3783] (cf. the ∼6 h needed for Boss-Sev to have an effect [2991]). In short, the pathway performs like a ﬁnely tuned (and easily tunable) musical instrument. Dos is also thought to bind Drk, dShc, PI3 kinase, and PLCγ in vivo [2639], but these liaisons are dispensable [243, 1830]. Dos is phosphorylated by Sev [1829] (Csw is not [71]), but whether Dos is likewise modiﬁed by Egfr is

304

APPENDIX SEVEN. COMMENTARIES ON THE PITHIER FIGURES

unclear. Apparently, Dos’s duty is to recruit Csw so that Csw can dephosphorylate Dos [1829] or a non-Dos target [1830]. In Torso signaling, Csw dephosphorylates Torso [808, 1403] so as to rebuff the SH2 domain of dRasGap (a Gap1-like protein) [808]. In Egfr and Sev signaling, Csw’s targets are unknown [808, 1403]: dephosphorylation may rebuff other dampers (e.g., PLCγ), and Csw’s chief role may be steric because it partially transmits a Sev signal even when catalytically inert [71]. Also murky is how Dos and dShc affect Ras1 or agents downstream. Whether dShc affects the pathway at Ras1 or farther downstream is unclear [392, 2623]. Other targets of dMAPK (links not shown) may include Sos and dMEK [2007]. Other phosphatases for dMAPK must exist aside from PTP-ER [2133] but are as yet unknown [2355] (cf. MKP-1 [4205] and MKP-3 [634, 1675, 2972], etc. [799, 2202, 2205, 3065]). Additional components remain to be identiﬁed (see text) [1237, 2532]. For further nuances, see [148, 381, 2957, 3899].

Figure 7.5 If Fz can localize asymmetrically within each cell in the eye as it does in the wing [4166], then the Vector Model would be favored. The identity of factor X is one of the great unsolved mysteries of ﬂy genetics [447, 2883, 4173, 4366]. Once it is found, we may be able to solve the general problem of how cells adopt particular orientations within epithelia: the “Planar Polarity Puzzle.” The robustness of this polarity mechanism in the eye (100% correct “read out” of the signal over a span of >100 cells) [2883, 4366] may stem from the superposition of multiple gradients with opposing slopes [4173, 4573]. A related mystery (albeit a morphogenetic one) is the “Gearing Riddle” [3563]. Namely, how do adjacent clusters rotate relative to one another? Some corollary issues are (1) how do outer cells make and break contacts with neighbors during the pivoting process?, (2) do the outer cells push or pull themselves along?, and (3) do they get traction by gripping their neighbors or by gaining purchase on a noncellular substratum? One clue is that solitary ommatidia can still rotate [181], so gearing per se may not be used. N.B.: Notch-dependent “symmetry breaking” is also thought to occur during selection of bristle SOPs (cf. Fig. 3.6) [3954]. Neither of the initial biasing events (Type 1 or 2) requires transcription, so they could happen fast. A precedent exists for Type-2 biasing in planar polarity: Starry night (a 7-pass cadherin; a.k.a. Flamingo) localizes to speciﬁc faces of wing cells at certain times in a Fz-dependent manner [447, 704, 4430]. Figure 7.6 By adding a long-range inhibitor (Argos) to its short-range activator (Spitz), the vocal cell reduces

the risk that the activator will mistakenly affect cells beyond its intended range (c, d). This trick for reducing the noise in sloppy signals is known as “lateral inhibition” [1479, 2806, 2815, 2818, 3207]. Argos apparently plays this same role in other tissues [1528, 3346, 3772, 4117] as well as possibly setting the intervals between ommatidial clusters (cf. Fig. 7.7) [4035]. In this regard, the nests of sensilla on the legs are intriguing. Those nests have constant numbers of sensilla trichodea (e.g., St8 on the coxa) or campaniformia (e.g., Sc11 on the femur) [526, 3705, 3807], and the number per nest can be changed by manipulating the Ras-MAPK pathway (L. Held, unpub. obs.). If Argos were to enable Spitz to behave like a membrane-bound ligand, then stepwise inductions via Spitz could “count” cells centrifugally in concentric rings around a founder cell – in effect computing a Fibonacci series based on the tiling geometries of the competent cells. The idea that induction merely “evokes” pre-existing abilities in responding cells is implicit in the Stop the Clock! Model. This same idea was endorsed long ago by Holtfreter [1885] and Waddington [4508]. N.B.: The peripodial membrane (omitted here) might play a role in photoreceptor patterning, but at present the evidence is only suggestive [773, 1473, 3508]. It may also be directing the odd vectors of regeneration that characterize this disc [3705]. The Stop the Clock! Model resembles the classic Clock and Wavefront Model, which was designed to explain the periodicity of somites in vertebrates [868, 4010]. In the latter model, the “Stop!” signal is conveyed by a wavefront instead of by juxtacrine signals [1805]. Surprisingly, vertebrate somitogenesis uses homologs of some of the ﬂy’s MF circuitry genes, including hairy [2224, 2484, 2988, 3236], Notch [856, 2051, 2061, 2095, 3451], and Delta [1878]. See [2786, 4111] for reviews, [3799, 4850] for related models, and [1181] for juxtacrine signaling in general.

Figure 7.7 The nuclei of disc cells migrate along the cell’s apical-basal axis during mitosis [20, 2650, 2744, 3422, 3426, 3539]. Curiously, R cells undergo similarly choreographed shifts during their differentiation [1742, 4355, 4364, 4715]. Such shifts were thought to be instrumental in cell signaling [533] or gene expression [2392, 3672], but they cannot be critical because differentiation proceeds fairly normally when the migrations are blocked [1239, 2483]. Perhaps, what matters here is the quiescence [604, 4387]. Cell cycle arrest is a general feature of proneural ﬁelds [2079, 4427, 4898], and the eye’s atonal stripe (Fig. 7.9) resembles AS-C stripes on the thorax and legs (cf. Figs. 3.8 and 3.9)

APPENDIX SEVEN. COMMENTARIES ON THE PITHIER FIGURES

[2020]. (Why evolution chose atonal for R8s and the AS-C for bristles is unknown [4897].) Quiescence might ensure precision in cell signaling (by homogenizing cell shapes? [287]) without affecting signals per se [1003, 2971, 3655]. Mitotic domains can be decisive in the embryo [110, 616, 1258, 3652, 4014], and wavefronts of various kinds seem to assign fates in other systems as well [1560, 1662, 1805]. This basic model was originally proposed not in ﬂies but in birds. In 1972, Donald Ede invented it to explain feather lattices in chicken skin [1140]. (Amazingly, bird feathers and ﬂy ommatidia may be patterned by similar gene cassettes [790, 1784, 2100, 3131, 3200, 3282, 3382, 4647].) Versions of this model were later advocated for the ﬂy eye [3574] using Sca [179, 2461, 2885], Argos [4035], or other molecules [204] as the inhibitor [1804, 2632]. N.B.: One quirk of the actual process (not shown) is that ommatidia arise ﬁrst in the equatorial portion of the MF (the “ﬁring center” [1638, 2631]), with later ones appearing at progressively more peripheral sites every ∼15 min. [182, 1076, 4712, 4873]. This sequential strategy avoids the stochastic errors (“stacking ﬂaws” [1639]) inherent in synchronous patterning (cf. Fig. 3.9c) and may thus ensure a greater uniformity in R8 intervals [3563], which are ∼7 cell diameters [4035, 4364]. Nevertheless, a problem arises at the MF’s outer tips, where new nodes do not have two prior nodes to delimit their “crevices” as the columns are getting taller [2632]. Some other device must help in this regard.

Figure 7.9 More genes undoubtedly remain to be incorporated into the circuit in b. Until they are found and subjected to LOF-GOF analysis, this puzzle will be unsolvable [2962]. Fortunately, future screens should be easier with clever new techniques that bypass the lethality of mutated vital genes [4132, 4292]. Genes omitted: eyelid (≈ hairy) [4389], m8 (≈ mδ) [229], orthodenticle (posterior to column 9) [4461], shortsighted (≈ hairy) [4388], Toll-like receptor (two stripes that “sandwich” the MF) [760], vein (weak expression in MF) [80]. Other genes are modulated in their expression along the A-P axis but show no sharp boundaries: Delta [186] and Notch [186]. Links not shown due to ambiguities: “hh hairy” anterior to the MF [1786, 3238, 4387] but “hh hairy” within

305

the MF (a threshold effect?) [1616], regulation of ato by Egfr (which is positive but has negative aftereffects) [4035], regulation of ato by Hh (which is positive or negative depending on the level of Hh) [1077], and regulation of ato by groucho and Hairless (which is also complex) [725]. Also not shown (to avoid clutter): “ato boss” [1076], “dpp hairy” [1616], “hh glass” [2632],“hh rough” [1077], and “hh sca” [2632, 3238]. Early (IC) and late (IG) expression of ato are controlled by separate cis-enhancers [4208]. For further details, see [182, 183, 720, 1076].

Figure 8.1 The BX-C is unusual in the vast amount of DNA that it devotes to cis-regulation (a) [1990, 2509]: only 1.4% of the locus actually encodes protein [2703] (cf. AS-C [1538], dpp [347, 2739, 4056], and homothorax [2360] loci). Note the similarity of the stages in b to the phases of information processing proposed in Wolpert’s Positional Information Hypothesis (cf. Fig. 4.3b). The 1987 “Open for Business Model” of Mark Peifer, Franc¸ois Karch, and Welcome Bender [3313] has been quite inﬂuential in this ﬁeld. It assumes that successively larger portions of the BX-C are available to trans-acting regulators in successively more posterior parasegments, but availability per se does not assure transcription. The relevant factors might be unevenly distributed within a parasegment, in which case Ubx, say, would be turned on nonuniformly (cf. Fig. 8.2). See [1770, 2304, 2305, 4165, 4719] for “nucleosome logic.” Figure 8.2 In essence, Ubx enables certain groups of cells in T3 discs to become distinct from the corresponding groups in T2 (i.e., it endows them with the competence to respond differently to the same ensemble of high-level “prepattern” regulators). Based on this one bit of difference (≈ “I am not my brother”), evolution has made various changes in the low-level “realizator” genes that control growth rates, pattern elements, etc. (≈ “I therefore do things differently”) [4563, 4564]. Interestingly, an analogous process occurred in the evolution of male vs. female sexual behavior. In that case, certain areas of the male brain were evolutionarily “painted” with the protein Fruitless, and they subseqently acquired circuits for the courtship of females [173]. (Pax6 in the eyes of arthropods vs. vertebrates is yet another example [3382].) What a difference one bit can make!

References

1. Aalfs, J.D. and Kingston, R.E. (2000). What does ‘chromatin remodeling’ mean? Trends Biochem. 25, 548–555. 2. Abbott, L.A. (1986). Restoration of wing margins in Lyra mutants of Drosophila melanogaster. Dev. Biol. 115, 233– 248. 3. Abbott, L.A. and Lindenmayer, A. (1981). Models for growth of clones in hexagonal cell arrangements: applications in Drosophila wing disc epithelia and plant epidermal tissues. J. Theor. Biol. 90, 495–544. 4. Abbott, L.A. and Sprey, T.E. (1990). Components of positional information in the developing wing margin of the Lyra mutant of Drosophila. Roux’s Arch. Dev. Biol. 198, 448–459. 5. Abbott, L.C., Karpen, G.H., and Schubiger, G. (1981). Compartmental restrictions and blastema formation during pattern regulation in Drosophila imaginal leg discs. Dev. Biol. 87, 64–75. 6. Abdelilah-Seyfried, S., Chan, Y.-M., Zeng, C., Justice, N.J., Younger-Shepherd, S., Sharp, L.E., Barbel, S., Meadows, S.A., Jan, L.Y., and Jan, Y.N. (2000). A gain-of-function screen for genes that affect the development of the Drosophila adult external sensory organ. Genetics 155, 733–752. 7. Aberle, H., Bauer, A., Stappert, J., Kispert, A., and Kemler, R. (1997). β-catenin is a target for the ubiquitin-proteasome pathway. EMBO J. 16, 3797–3804. 8. Abu-Shaar, M. and Mann, R.S. (1998). Generation of multiple antagonistic domains along the proximodistal axis during Drosophila leg development. Development 125, 3821–3830. 9. Abu-Shaar, M., Ryoo, H.D., and Mann, R.S. (1999). Control of the nuclear localization of Extradenticle by competing nuclear import and export signals. Genes Dev. 13, 935–945. 10. Abzhanov, A. and Kaufman, T.C. (2000). Homologs of Drosophila appendage genes in the patterning of arthropod limbs. Dev. Biol. 227, 673–689. 11. Adachi, M., Fukuda, M., and Nishida, E. (1999). Two coexisting mechanisms for nuclear import of MAP kinase: passive diffusion of a monomer and active transport of a dimer. EMBO J. 18, 5347–5358. 12. Adachi-Yamada, T., Fujimura-Kamada, K., Nishida, Y., and Matsumoto, K. (1999). Distortion of proximodistal infor-

mation causes JNK-dependent apoptosis in Drosophila wing. Nature 400, 166–169. 13. Adachi-Yamada, T., Nakamura, M., Irie, K., Tomoyasu, Y., Sano, Y., Mori, E., Goto, S., Ueno, N., Nishida, Y., and Matsumoto, K. (1999). p38 mitogen-activated protein kinase can be involved in Transforming Growth Factor β superfamily signal transduction in Drosophila wing morphogenesis. Mol. Cell. Biol. 19, 2322–2329. 14. Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., Gocayne, J.D., Amanatides, P.G., Scherer, S.E., Li, P.W., Hoskins, R.A., Galle, R.F., George, R.A., Lewis, S.E., Richards, S., Ashburner, M., Henderson, S.N., Sutton, G.G., Wortman, J.R., Yandell, M.D., Zhang, Q., Chen, L.X., Brandon, R.C., Rogers, Y.-H.C., Blazej, R.G., Champe, M., Pfeiffer, B.D., Wan, K.H., Doyle, C., Baxter, E.G., Helt, G., Nelson, C.R., Gabor Miklos, G.L., Abril, J.F., Agbayani, A., An, H.-J., Andrews-Pfannkoch, C.A., Baldwin, D., Ballew, R.M., Basu, A., Baxendale, J., Bayraktaroglu, L., Beasley, E.M., Beeson, K.Y., Benos, P.V., Berman, B.P., Bhandari, D., Bolshakov, S., Borkova, D., Botchan, M.R., Bouck, J., Brokstein, P., Brottier, P., Burtis, K.C., Busam, D.A., Butler, H., Cadieu, E., Center, A., Chandra, I., Cherry, J.M., Cawley, S., Dahlke, C., Davenport, L.B., Davies, P., de Pablos, B., Delcher, A., Deng, Z., Deslattes Mays, A., Dew, I., Dietz, S.M., Dodson, K., Doup, L.E., Downes, M., DuganRocha, S., Dunkov, B.C., Dunn, P., Durbin, K.J., Evangelista, C.C., Ferraz, C., Ferriera, S., Fleischmann, W., Fosler, C., Gabrielian, A.E., Garg, N.S., Gelbart, W.M., Glasser, K., Glodek, A., Gong, F., Gorrell, J.H., Gu, Z., Guan, P., Harris, M., Harris, N.L., Harvey, D., Heiman, T.J., Hernandez, J.R., Houck, J., Hostin, D., Houston, K.A., Howland, T.J., Wei, M.-H., Ibegwam, C., Jalali, M., Kalush, F., Karpen, G.H., Ke, Z., Kennison, J.A., Ketchum, K.A., Kimmel, B.E., Kodira, C.D., Kraft, C., Kravitz, S., Kulp, D., Lai, Z., Lasko, P., Lei, Y., Levitsky, A.A., Li, J., Li, Z., Liang, Y., Lin, X., Liu, X., Mattei, B., McIntosh, T.C., McLeod, M.P., McPherson, D., Merkulov, G., Milshina, N.V., Mobarry, C., Morris, J., Moshreﬁ, A., Mount, S.M., Moy, M., Murphy, B., Murphy, L., Muzny, D.M., Nelson, D.L., Nelson, D.R., Nelson, K.A., Nixon, K., Nusskern, D.R., Pacleb, J.M., Palazzolo, M., Pittman, G.S., Pan, S., Pollard, J., Puri, V., Reese, M.G., Reinert, K., Remington, K., Saunders, R.D.C., Scheeler,

307

308

15.

16.

17.

18.

19. 20.

21.

22.

23.

24. 25.

26.

27.

28.

29.

30.

REFERENCES

F., Shen, H., Shue, B.C., Sid´en-Kiamos, I., Simpson, M., Skupski, M.P., Smith, T., Spier, E., Spradling, A.C., Stapleton, M., Strong, R., Sun, E., Svirskas, R., Tector, C., Turner, R., Venter, E., Wang, A.H., Wang, X., Wang, Z.-Y., Wassarman, D.A., Weinstock, G.M., Weissenbach, J., Williams, S.M., Woodage, T., Worley, K.C., Wu, D., Yang, S., Yao, Q.A., Ye, J., Yeh, R.-F., Zaveri, J.S., Zhan, M., Zhang, G., Zhao, Q., Zheng, L., Zheng, X.H., Zhong, F.N., Zhong, W., Zhou, X., Zhu, S., Zhu, X., Smith, H.O., Gibbs, R.A., Myers, E.W., Rubin, G.M. and Venter, J.C. (2000). The genome sequence of Drosophila melanogaster. Science 287, 2185– 2195. [See also 2001 Nature 411, 259–260.] Adamson, A.L. and Shearn, A. (1996). Molecular genetic analysis of Drosophila ash2, a member of the trithorax group required for imaginal disc pattern formation. Genetics 144, 621–633. Adler, P. (1978). Mutants of the Bithorax Complex and determinative states in the thorax of Drosophila melanogaster. Dev. Biol. 65, 447–461. Adler, P.N. (1978). Positional information in imaginal discs transformed by homoeotic mutations. Fate map and regulative behavior of fragments of haltere discs transformed by bithorax 3 and postbithorax. W. Roux’s Arch. 185, 271– 292. Adler, P.N. (1979). Position-speciﬁc interaction between cells of the imaginal wing and haltere discs of Drosophila melanogaster. Dev. Biol. 70, 262–267. Adler, P.N. (1981). Growth during pattern regulation in imaginal discs. Dev. Biol. 87, 356–373. Adler, P.N. (1987). Growth and pattern regulation in insects. In “Invertebrate Models: Cell Receptors and Cell Communication,” (A.H. Greenberg, ed.). Karger: Basel, pp. 172–189. Adler, P.N., Charlton, J., and Liu, J. (1998). Mutations in the cadherin superfamily member gene dachsous cause a tissue polarity phenotype by altering frizzled signaling. Development 125, 959–968. Adler, P.N., Krasnow, R.E., and Liu, J. (1997). Tissue polarity points from cells that have higher Frizzled levels towards cells that have lower Frizzled levels. Curr. Biol. 7, 940–949. Adler, P.N. and MacQueen, M. (1981). Partial coupling of the cell cycles of neighboring imaginal disc cells. Exp. Cell Res. 133, 452–456. Adler, P.N. and Taylor, J. (2001). Asymmetric cell division: plane but not simple. Curr. Biol. 11, R233–R236. Adler, P.N., Taylor, J., and Charlton, J. (2000). The domineering non-autonomy of frizzled and Van Gogh clones in the Drosophila wing is a consequence of a disruption in local signaling. Mechs. Dev. 96, 197–207. Adler, P.N., Vinson, C., Park, W.J., Conover, S., and Klein, L. (1990). Molecular structure of frizzled, a Drosophila tissue polarity gene. Genetics 126, 401–416. Adler, R. and Hatlee, M. (1989). Plasticity and differentiation of embryonic retinal cells after terminal mitosis. Science 243, 391–393. Affolter, M., Marty, T., and Vigano, M.A. (1999). Balancing import and export in development. Genes Dev. 13, 913– 915. Affolter, M., Montagne, J., Walldorf, U., Groppe, J., Kloter, U., LaRosa, M., and Gehring, W.J. (1994). The Drosophila SRF homolog is expressed in a subset of tracheal cells and maps within a genomic region required for tracheal development. Development 120, 743–753. Affolter, M., Nellen, D., Nussbaumer, U., and Basler, K.

31.

32. 33.

34.

35.

36. 37.

38.

39.

40. 41. 42. 43.

44.

45. 46. 47. 48. 49. 50. 51.

(1994). Multiple requirements for the receptor serine/ threonine kinase thick veins reveal novel functions of TGFβ homologs during Drosophila embryogenesis. Development 120, 3105–3117. ¨ Affolter, M., Percival-Smith, A., Muller, M., Billeter, M., ¨ Qian, Y.Q., Otting, G., Wuthrich, K., and Gehring, W.J. (1991). Similarities between the homeodomain and the Hin recombinase DNA-binding domain. Cell 64, 879–880. Agarwal, P. (1995). The Cell Programming Language. Artif. Life 2, 37–77. Agnel, M., R¨oder, L., Grifﬁn-Shea, R., and Vola, C. (1992). The spatial expression of Drosophila rotund gene reveals that the imaginal discs are organized in domains along the proximal-distal axis. Roux’s Arch. Dev. Biol. 201, 284–295. Agn`es, F., Suzanne, M., and Noselli, S. (1999). The Drosophila JNK pathway controls the morphogenesis of imaginal discs during metamorphosis. Development 126, 5453– 5462. Agrawal, N., Kango, M., Mishra, A., and Sinha, P. (1995). Neoplastic transformation and aberrant cell-cell interactions in genetic mosaics of lethal(2)giant larvae (lgl), a tumor suppressor gene of Drosophila. Dev. Biol. 172, 218– 229. Agutter, P.S. and Wheatley, D.N. (2000). Random walks and cell size. BioEssays 22, 1018–1023. Ahmad, K. and Henikoff, S. (2001). Modulation of transcription factor counteracts heterochromatic gene silencing in Drosophila. Cell 104, 839–847. Ahmad, S.M. and Baker, B.S. (2001). Sex-speciﬁc deployment of FGF-signalling in Drosophila recruits mesodermal cells into the male genital imaginal disc. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a49. Ahmed, Y., Hayashi, S., Levine, A., and Wieschaus, E. (1998). Regulation of Armadillo by a Drosophila APC inhibits neuronal apoptosis during retinal development. Cell 93, 1171–1182. Ahrendt, W.R. and Savant, C.J., Jr. (1960). “Servomechanism Practice.” McGraw-Hill, New York. Ahringer, J. (2000). NuRD and SIN3: histone deacetylase complexes in development. Trends Genet. 16, 351–356. Aitken, A. (1995). 14-3-3 proteins on the MAP. Trends Biochem. Sci. 20, 95–97. Aitken, A. (1996). 14-3-3 and its possible role in coordinating multiple signalling pathways. Trends Cell Biol. 6, 341–347. Aitken, A., Collinge, D.B., van Heusden, B.P.H., Isobe, T., Roseboom, P.H., Rosenfeld, G., and Soll, J. (1992). 14-3-3 proteins: a highly conserved, widespread family of eukaryotic proteins. Trends Biochem. Sci. 17, 498–501. [See also 2001 BioEssays 23, 936–946.] Akam, M. (1984). A common segment in genes for segments of Drosophila. Nature 308, 402–403. Akam, M. (1984). A molecular theme for homoeotic genes. BioEssays 1, 78–79. Akam, M. (1987). The molecular basis for metameric pattern in the Drosophila embryo. Development 101, 1–22. Akam, M. (1989). Hox and HOM: homologous gene clusters in insects and vertebrates. Cell 57, 347–349. Akam, M. (1989). Making stripes inelegantly. Nature 341, 282–283. Akam, M. (1991). Wondrous transformation. Nature 349, 282. Akam, M. (1998). Hox genes: From master genes to micromanagers. Curr. Biol. 8, R676–R678.

REFERENCES

52. Akam, M. (1998). Hox genes, homeosis and the evolution of segment identity: no need for hopeless monsters. Int. J. Dev. Biol. 42, 445–451. 53. Akam, M.E. and Martinez-Arias, A. (1985). The distribution of Ultrabithorax transcripts in Drosophila embryos. EMBO J. 4, 1689–1700. 54. Akimaru, H., Chen, Y., Dai, P., Hou, D.-X., Nonaka, M., Smolik, S.M., Armstrong, S., Goodman, R.H., and Ishii, S. (1997). Drosophila CBP is a co-activator of cubitus interruptus in hedgehog signalling. Nature 386, 735–738. 55. Alber, T. (1993). How GCN4 binds DNA. Curr. Biol. 3, 182– 184. 56. Alberch, P. (1982). Developmental constraints in evolutionary processes. In “Evolution and Development,” (J.T. Bonner, ed.). Springer-Verlag: Berlin, pp. 313–332. 57. Alberga, A., Boulay, J.-L., Kempe, E., Dennefeld, C., and Haenlin, M. (1991). The snail gene required for mesoderm formation in Drosophila is expressed dynamically in derivatives of all three germ layers. Development 111, 983–992. 58. Alberts, B. (1998). The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 92, 291–294. 59. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J.D. (1994). “Molecular Biology of the Cell.” 3rd ed. Garland, New York. 60. Albright, T.D., Jessell, T.M., Kandel, E.R., and Posner, M.I. (2000). Neural science: a century of progress and the mysteries that remain. Cell 100 (Suppl.), S1–S55. 61. Alcedo, J., Ayzenzon, M., Von Ohlen, T., Noll, M., and Hooper, J.E. (1996). The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the Hedgehog signal. Cell 86, 221–232. 62. Alcedo, J., Zou, Y., and Noll, M. (2000). Posttranscriptional regulation of Smoothened is part of a self-correcting mechanism in the Hedgehog signaling system. Molec. Cell 6, 457–465. 63. Alevizopoulos, A. and Mermod, N. (1997). Transforming growth factor-β: the breaking open of a black box. BioEssays 19, 581–591. 64. Alexander, R.M. (1995). Big ﬂies have bigger cells. Nature 375, 20. 65. Alexandre, C., Jacinto, A., and Ingham, P.W. (1996). Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the Cubitus interruptus protein, a member of the GLI family of zinc ﬁnger DNAbinding proteins. Genes Dev. 10, 2003–2013. 66. Alexandre, E., Graba, Y., Fasano, L., Gallet, A., L., P., De Zulueta, P., Pradel, J., Kerridge, S., and Jacq, B. (1996). The Drosophila Teashirt homeotic protein is a DNA-binding protein and modulo, a HOM-C regulated modiﬁer of variegation, is a likely candidate for being a direct target gene. Mechs. Dev. 59, 191–204. 67. Ali, Z., Drees, B., Coleman, K.G., Gustavson, E., Karr, T.L., Kauvar, L.M., Poole, S.J., Soeller, W., Weir, M.P., and Kornberg, T. (1985). The engrailed locus of Drosophila melanogaster : genetic, developmental, and molecular studies. Cold Spr. Harb. Symp. Quant. Biol. 50, 229–233. 68. Alifragis, P., Poortinga, G., Parkhurst, S.M., and Delidakis, C. (1997). A network of interacting transcriptional regulators involved in Drosophila neural fate speciﬁcation revealed by the yeast two-hybrid system. Proc. Natl. Acad. Sci. USA 94, 13099–13104. 69. Allada, R., White, N.E., So, W.V., Hall, J.C., and Rosbash,

309

70.

71.

72. 73.

74. 75.

76.

77.

78.

79.

80.

81.

82.

83. 84.

85.

86. 87.

M. (1998). A mutant Drosophila homolog of mammalian Clock disrupts circadian rhythms and transcription of period and timeless. Cell 93, 791–804. Allard, J.D., Chang, H.C., Herbst, R., McNeill, H., and Simon, M.A. (1996). The SH2-containing tyrosine phosphatase corkscrew is required during signaling by sevenless, Ras1 and Raf. Development 122, 1137–1146. Allard, J.D., Herbst, R., Carroll, P.M., and Simon, M.A. (1998). Mutational analysis of the SRC homology 2 domain protein-tyrosine phosphatase Corkscrew. J. Biol. Chem. 273 #21, 13129–13135. Allen, G.E. (1978). “Thomas Hunt Morgan: The Man and His Science.” Princeton Univ. Pr., Princeton, N. J. Alonso, M.C. and Cabrera, C.V. (1988). The achaete-scute gene complex of Drosophila melanogaster comprises four homologous genes. EMBO J. 7, 2585–2591. Alpert, M. (1999). Not just fun and games. Sci. Am. 280 #4, 40–42. Alton, A.K., Fechtel, K., Kopczynski, C.C., Shepard, S.B., Kooh, P.J., and Muskavitch, M.A.T. (1989). Molecular genetics of Delta, a locus required for ectodermal differentiation in Drosophila. Dev. Genet. 10, 261–272. Alton, A.K., Fechtel, K., Terry, A.L., Meikle, S.B., and Muskavitch, M.A.T. (1988). Cytogenetic deﬁnition and morphogenetic analysis of Delta, a gene affecting neurogenesis in Drosophila melanogaster. Genetics 118, 235– 245. Alvarado, D. and Duffy, J.B. (2001). Functional characterization of KEKKON1 during Drosophila oogenesis. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a167. Alves, G., Limbourg-Bouchon, B., Tricoire, H., BrissardZahraoui, J., Lamour-Isnard, C., and Busson, D. (1998). Modulation of Hedgehog target gene expression by the Fused serine-threonine kinase in wing imaginal discs. Mechs. Dev. 78, 17–31. Ambros, V. (1999). Cell cycle-dependent sequencing of cell fate decisions in Caenorhabditis elegans vulva precursor cells. Development 126, 1947–1956. Amin, A., Li, Y., and Finkelstein, R. (1999). Hedgehog activates the EGF receptor pathway during Drosophila head development. Development 126, 2623–2630. Anderson, D.T. (1963). The embryology of Dacus tryoni. 2. Development of imaginal discs in the embryo. J. Embryol. Exp. Morph. 11, 339–351. Anderson, D.T. (1972). The development of holometabolous insects. In “Developmental Systems: Insects,” Vol. 1 (S.J. Counce and C.H. Waddington, eds.). Acad. Pr.: New York, pp. 165–242. [See also 2001 Evol. Dev. 3, 59–72.] Anderson, J.M. (1996). Cell signalling: MAGUK magic. Curr. Biol. 6, 382–384. Anderson, M.G., Certel, S.J., Certel, K., Lee, T., Montell, D.J., and Johnson, W.A. (1996). Function of the Drosophila POU domain transcription factor Drifter as an upstream regulator of Breathless receptor tyrosine kinase expression in developing trachea. Development 122, 4169–4178. Anderson, M.G., Perkins, G.L., Chittick, P., Shrigley, R.J., and Johnson, W.A. (1995). drifter, a Drosophila POUdomain transcription factor, is required for correct differentiation and migration of tracheal cells and midline glia. Genes Dev. 9, 123–137. Anderton, B.H. (1999). Alzheimer’s disease: clues from ﬂies and worms. Curr. Biol. 9, R106–R109. Andr´es, V., Chiara, M.D., and Mahdavi, V. (1994). A new bipartite DNA-binding domain: cooperative interaction

310

88.

89.

90. 91.

92.

93.

94.

95. 96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

REFERENCES

between the cut repeat and homeo domain of the cut homeo proteins. Genes Dev. 8, 245–257. Andrew, D.J., Baig, A., Bhanot, P., Smolik, S.M., and Henderson, K.D. (1997). The Drosophila dCREB-A gene is required for dorsal/ventral patterning of the larval cuticle. Development 124, 181–193. Andrew, D.J., Horner, M.A., Petitt, M.G., Smolik, S.M., and Scott, M.P. (1994). Setting limits on homeotic gene function: restraint of Sex combs reduced activitiy by teashirt and other homeotic genes. EMBO J. 13, 1132–1144. Andrews, D.W. (2000). Transport across membranes: a question of navigation. Cell 102, 139–144. Angerer, L.M. and Angerer, R.C. (1999). Regulative development of the sea urchin embryo: Signalling cascades and morphogen gradients. Sems. Cell Dev. Biol. 10, 327– 334. Apidianakis, Y., Nagel, A.C., Chalkiadaki, A., Preiss, A., and Delidakis, C. (1999). Overexpression of the m4 and mα genes of the E(spl)-Complex antagonizes Notch mediated lateral inhibition. Mechs. Dev. 86, 39–50. Aplin, A.C. and Kaufman, T.C. (1997). Homeotic transformation of legs to mouthparts by proboscipedia expression in Drosophila imaginal discs. Mechs. Dev. 62, 51–60. Appel, L.F., Prout, M., Abu-Shumays, R., Hammonds, A., Garbe, J.C., Fristrom, D., and Fristrom, J. (1993). The Drosophila Stubble-stubbloid gene encodes an apparent transmembrane serine protease required for epithelial morphogenesis. Proc. Natl. Acad. Sci. USA 90, 4937–4941. Apter, M.J. and Wolpert, L. (1965). Cybernetics and development. I. Information theory. J. Theor. Biol. 8, 244–257. Araujo, H. and Bier, E. (2000). sog and dpp exert opposing maternal functions to modify Toll signaling and pattern the dorsoventral axis of the Drosophila embryo. Development 127, 3631–3644. Arber, S. and Caroni, P. (1996). Speciﬁcity of single LIM motifs in targeting and LIM/LIM interactions in situ. Genes Dev. 10, 289–300. Arbib, M.A. (1972). Automata theory in the context of theoretical embryology. In “Foundations of Mathematical Biology,” Vol. 2 (R. Rosen, ed.). Acad. Pr.: New York, pp. 141–215. Argyris, T.S. (1972). Chalones and the control of normal, regenerative, and neoplastic growth of the skin. Amer. Zool. 12, 137–149. Arias, J., Alberts, A.S., Brindle, P., Claret, F.X., Smeal, T., Karin, M., Feramisco, J., and Montminy, M. (1994). Activation of cAMP and mitogen responsive genes relies on a common nuclear factor. Nature 370, 226–229. Arking, R. (1975). Temperature-sensitive cell-lethal mutants of Drosophila: isolation and characterization. Genetics 80, 519–537. Arnheim, N., Jr. (1967). The regional effects of two mutants in Drosophila analyzed by means of mosaics. Genetics 56, 253–263. Arnone, M.I. and Davidson, E.H. (1997). The hardwiring of development: organization and function of genomic regulatory systems. Development 124, 1851–1864. Arnosti, D.N., Barolo, S., Levine, M., and Small, S. (1996). The eve stripe 2 enhancer employs multiple modes of transcriptional synergy. Development 122, 205–214. Arnosti, D.N., Gray, S., Barolo, S., Zhou, J., and Levine, M. (1996). The gap protein knirps mediates both quenching and direct repression in the Drosophila embryo. EMBO J. 15, 3659–3666.

106. Aronheim, A., Engelberg, D., Li, N., Al-Alawi, N., Schlessinger, J., and Karin, M. (1994). Membrane targeting of the nucleotide exchange factor Sos is sufﬁcient for activating the Ras signaling pathway. Cell 78, 949–961. 107. Aronson, B.D., Fisher, A.L., Blechman, K., Caudy, M., and Gergen, J.P. (1997). Groucho-dependent and -independent repression activities of Runt domain proteins. Mol. Cell. Biol. 17, 5581–5587. 108. Arora, K., Dai, H., Kazuko, S.G., Jamal, J., O’Connor, M.B., Letsou, A., and Warrior, R. (1995). The Drosophila schnurri gene acts in the Dpp/TGFβ signaling pathway and encodes a transcription factor homologous to the human MBP family. Cell 81, 781–790. 109. Arora, K., Levine, M.S., and O’Connor, M.B. (1994). The screw gene encodes a ubiquitously expressed member of the TGF-β family required for speciﬁcation of dorsal cell fates in the Drosophila embryo. Genes Dev. 8, 2588–2601. ¨ 110. Arora, K. and Nusslein-Volhard, C. (1992). Altered mitotic domains reveal fate map changes in Drosophila embryos mutant for zygotic dorsoventral patterning genes. Development 114, 1003–1024. 111. Arora, K., Rodrigues, V., Joshi, S., Shanbhag, S., and Siddiqi, O. (1987). A gene affecting the speciﬁcity of the chemosensory neurons of Drosophila. Nature 330, 62–63. 112. Artavanis-Tsakonas, S., Matsuno, K., and Fortini, M.E. (1995). Notch signaling. Science 268, 225–232. 113. Artavanis-Tsakonas, S., Rand, M.D., and Lake, R.J. (1999). Notch signaling: cell fate control and signal integration in development. Science 284, 770–776. 114. Artavanis-Tsakonas, S. and Simpson, P. (1991). Choosing a cell fate: a view from the Notch locus. Trends Genet. 7, 403–408. 115. Artero, R.D., Akam, M., and P´erez-Alonso, M. (1992). Oligonucleotide probes detect splicing variants in situ in Drosophila embryos. Nucl. Acids Res. 20, 5687–5690. 116. Ascano, M., Nybakken, K.E., Vallance, J.E., and Robbins, D.J. (2001). Disruption of a Hedgehog signaling complex. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a160. 117. Ashburner, M. (1982). The genetics of a small autosomal region of Drosophila melanogaster containing the structural gene for alcohol dehydrogenase. III. Hypomorphic and hypermorphic mutations affecting the expression of Hairless. Genetics 101, 447–459. 118. Ashburner, M. (1989). “Drosophila: A Laboratory Handbook.” CSH Pr., Cold Spring Harbor, N.Y. 119. Ashburner, M. (1993). Epilogue. In “The Development of Drosophila melanogaster,” Vol. 2 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Lab. Pr.: Plainview, N. Y., pp. 1493–1506. 120. Ashburner, M. (1995). Maps and legends. Trends Genet. 11, 33–34. 121. Ashburner, M. and Berendes, H.D. (1978). Pufﬁng of polytene chromosomes. In “The Genetics and Biology of Drosophila,” Vol. 2b (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 315–395. 122. Ashburner, M., Carson, H.L., Novitski, E., Thompson, J.N., and Wright, T.R.F., eds. (1976–1986). “The Genetics and Biology of Drosophila.” Vol. 1 (a-c), 2 (a-d), 3 (a-e). Acad. Pr., London. 123. Ashburner, M., Chihara, C., Meltzer, P., and Richards, G. (1974). Temporal control of pufﬁng activity in polytene chromosomes. Cold Spr. Harb. Symp. Quant. Biol. 38, 655– 662. 124. Ashburner, M. and Drysdale, R. (1994). FlyBase–The

REFERENCES

125. 126.

127.

128.

129.

130.

131.

132.

133.

134.

135.

136. 137. 138. 139.

140.

141.

Drosophila genetic database. Development 120, 2077– 2079. Ashburner, M. and Lawrence, P. (1988). Fruitful fruitﬂies in Crete. Nature 336, 620–621. Ashburner, M., Tsubota, S., and Woodruff, R.C. (1982). The genetics of a small chromosome region of Drosophila melanogaster containing the structural gene for alcohol dehydrogenase. IV. Scutoid, an antimorphic mutation. Genetics 102, 401–420. Ashe, H.L., Mannervik, M., and Levine, M. (2000). Dpp signaling thresholds in the dorsal ectoderm of the Drosophila embryo. Development 127, 3305–3312. Ashley, J.A. and Katz, F.N. (1994). Competition and position-dependent targeting in the development of the Drosophila R7 visual projections. Development 120, 1537– 1547. Aslam, H. and Baron, M. (2001). Suppressor of deltex requires a ubiquitin ligase domain to downregulate the Notch signal. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a168. Aspland, S.E. and White, R.A.H. (1997). Nucleocytoplasmic localisation of extradenticle protein is spatially regulated throughout development in Drosophila. Development 124, 741–747. Assa-Munt, N., Mortishire-Smith, R.J., Aurora, R., Herr, W., and Wright, P.E. (1993). The solution structure of the Oct-1 POU-speciﬁc domain reveals a striking similarity to the bacteriophage l repressor DNA-binding domain. Cell 73, 193–205. Aster, J.C., Robertson, E.S., Hasserjian, R.P., Turner, J.R., Kieff, E., and Sklar, J. (1997). Oncogenic forms of NOTCH1 lacking either the primary binding site for RBP-Jκ or nuclear localization sequences retain the ability to associate with RBP-Jκ and activate transcription. J. Biol. Chem. 272 #17, 11336–11343. Astor, J.C. and Adami, C. (2001). A developmental model for the evolution of artiﬁcial neural networks. Artif. Life 6, 189–218. Asturias, F.J., Jiang, Y.W., Myers, L.C., Gustafsson, C.M., and Kornberg, R.D. (1999). Conserved structures of mediator and RNA polymerase II holoenzyme. Science 283, 985–987. Atchley, W.R. and Fitch, W.M. (1997). A natural classiﬁcation of the basic helix-loop-helix class of transcription factors. Proc. Natl. Acad. Sci. USA 94, 5172–5176. Atlan, H. and Koppel, M. (1990). The cellular computer DNA: program or data. Bull. Math. Biol. 52, 335–348. Attisano, L. and Wrana, J.L. (1998). Mads and Smads in TGFβ signalling. Curr. Opin. Cell Biol. 10, 188–194. Attisano, L. and Wrana, J.L. (2000). Smads as transcriptional co-modulators. Curr. Opin. Cell Biol. 12, 235–243. Attisano, L., Wrana, J.L., L´opez-Casillas, F., and Massagu´e, J. (1994). TGF-β receptors and actions. Bioch. Biophys. Acta 1222, 71–80. Audibert, A., Juge, F., and Simonelig, M. (1998). The suppressor of forked protein of Drosophila, a homologue of the human 77K protein required for mRNA 3 -end formation, accumulates in mitotically-active cells. Mechs. Dev. 72, 53–63. Audibert, A. and Simonelig, M. (1999). The suppressor of forked gene of Drosophila, which encodes a homologue of human CstF-77K involved in mRNA 3 -end processing, is required for progression through mitosis. Mechs. Dev. 82, 41–50.

311

142. Auerbach, C. (1936). The development of the legs, wings, and halteres in wild type and some mutant strains of Drosophila melanogaster. Trans. Roy. Soc. Edin. 58, 787– 815. 143. Avedisov, S.N., Krasnoselskaya, I., Mortin, M., and Thomas, B.J. (2000). Roughex mediates G1 arrest through a physical association with Cyclin A. Mol. Cell. Biol. 20, 8220–8229. 144. Averof, M. (1997). Arthropod evolution: Same Hox genes, different body plans. Curr. Biol. 7, R634–R636. 145. Averof, M. and Cohen, S.M. (1997). Evolutionary origin of insect wings from ancestral gills. Nature 385, 627–630. 146. Averof, M., Dawes, R., and Ferrier, D. (1996). Diversiﬁcation of arthropod Hox genes as a paradigm for the evolution of gene functions. Sems. Cell Dev. Biol. 7, 539–551. 147. Avery, L. and Wasserman, S. (1992). Ordering gene function: the interpretation of epistasis in regulatory hierarchies. Trends Genet. 8, 312–316. 148. Avruch, J., Zhang, X.-f., and Kyriakis, J.M. (1994). Raf meets Ras: completing the framework of a signal transduction pathway. Trends Biochem. Sci. 19, 279–284. 149. Awasaki, T. and Kimura, K.-i. (1997). pox-neuro is required for development of chemosensory bristles in Drosophila. J. Neurobiol. 32, 707–721. 150. Awasaki, T. and Kimura, K.-i. (2001). Multiple function of poxn gene in larval PNS development and in adult appendage formation of Drosophila. Dev. Genes Evol. 211, 20–29. 151. Axelrod, J.D., Matsuno, K., Artavanis-Tsakonas, S., and Perrimon, N. (1996). Interaction between Wingless and Notch signaling pathways mediated by Dishevelled. Science 271, 1826–1832. [See also 2001 Curr. Biol. 11, 1729–1738.] 152. Axelrod, J.D., Miller, J.R., Shulman, J.M., Moon, R.T., and Perrimon, N. (1998). Differential recruitment of Dishevelled provides signaling speciﬁcity in the planar cell polarity and Wingless signaling pathways. Genes Dev. 12, 2610– 2622. 153. Ayyar, S. and White, R.A.H. (2001). achintya and vismay : two novel, tandem-repeated TALE-class homeobox genes. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a177. 154. Aza-Blanc, P. and Kornberg, T.B. (1999). Ci: a complex transducer of the Hedgehog signal. Trends Genet. 15, 458– 462. 155. Aza-Blanc, P., Ram´ırez-Weber, F.-A., Laget, M.-P., Schwartz, C., and Kornberg, T.B. (1997). Proteolysis that is inhibited by Hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell 89, 1043–1053. 156. Azpiazu, N. and Morata, G. (1998). Functional and regulatory interactions between Hox and extradenticle genes. Genes Dev. 12, 261–273. 157. Azpiazu, N. and Morata, G. (2000). Function and regulation of homothorax in the wing imaginal disc of Drosophila. Development 127, 2685–2693. 158. Babu, P. (1977). Early developmental subdivisions of the wing disk in Drosophila. Mol. Gen. Genet. 151, 289–294. 159. Babu, P. and Bhat, S.G. (1986). Autonomy of the wingless mutation in Drosophila melanogaster. Mol. Gen. Genet. 205, 483–486. 160. Bach, I. (2000). The LIM domain: regulation by association. Mechs. Dev. 91, 5–17. 161. Bach, I., Rodriguez-Esteban, C., Carri´ere, C., Bhushan, A., ´ Krones, A., Rose, D.W., Glass, C.K., Andersen, B., Izpisua

312

162.

163.

164.

165.

166.

167.

168. 169.

170.

171.

172.

173.

174. 175.

176.

177.

178. 179.

REFERENCES

Belmonte, J.C., and Rosenfeld, M.G. (1999). RLIM inhibits functional activity of LIM homeodomain transcription factors via recruitment of the histone deacetylase complex. Nature Genet. 22, 394–399. Bachiller, D., Mac´ıas, A., Duboule, D., and Morata, G. (1994). Conservation of a functional hierarchy between mammalian and insect Hox/HOM genes. EMBO J. 13, 1930–1941. Bachmann, A. and Knust, E. (1998). Dissection of cisregulatory elements of the Drosophila gene Serrate. Dev. Genes Evol. 208, 346–351. Bachmann, A. and Knust, E. (1998). Positive and negative control of Serrate expression during early development of the Drosophila wing. Mechs. Dev. 76, 67–78. Bae, K., Lee, C., Sidote, D., Chuang, K.-Y., and Edery, I. (1998). Circadian regulation of a Drosophila homolog of the mammalian Clock gene: PER and TIM function as positive regulators. Mol. Cell. Biol. 18, 6142–6151. Baeg, G.-H., Lin, X., Khare, N., Baumgartner, S., and Perrimon, N. (2001). Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development 128, 87–94. Baek, K.-H., Fabian, J.R., Sprenger, F., Morrison, D.K., and Ambrosio, L. (1996). The activity of D-raf in Torso signal transduction is altered by serine substitution, N-terminal deletion and membrane targeting. Dev. Biol. 175, 191–204. Baeuerle, P.A. (1998). IκB-NF-κB structures: At the interface of inﬂammation control. Cell 95, 729–731. Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J.W., and Elledge, S.J. (1996). SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86, 263–274. Bai, J.-M., Chiu, W.-H., Wang, J.-C., Tzeng, T.-H., Perrimon, N., and Hsu, J.-C. (2001). The cell adhesion molecule Echinoid deﬁnes a new pathway that antagonizes the Drosophila EGF receptor signaling pathway. Development 128, 591–601. Bailey, A.M. and Posakony, J.W. (1995). Suppressor of Hairless directly activates transcription of Enhancer of split Complex genes in response to Notch receptor activity. Genes Dev. 9, 2609–2622. Baker, A., Kaplan, C.P., and Pool, M.R. (1996). Protein targeting and translocation; a comparative survey. Biol. Rev. 71, 637–702. Baker, B.S., Taylor, B.J., and Hall, J.C. (2001). Are complex behaviors speciﬁed by dedicated regulatory genes? Reasoning from Drosophila. Cell 105, 13–24. Baker, J.C. and Harland, R.M. (1997). From receptor to nucleus: the Smad pathway. Curr. Opin. Gen. Dev. 7, 467–473. Baker, N.E. (1987). Molecular cloning of sequences from wingless, a segment polarity gene in Drosophila: the spatial distribution of a transcript in embryos. EMBO J. 6, 1765–1773. Baker, N.E. (1988). Localization of transcripts from the wingless gene in whole Drosophila embryos. Development 103, 289–298. Baker, N.E. (1988). Transcription of the segment-polarity gene wingless in the imaginal discs of Drosophila, and the phenotype of a pupal-lethal wg mutation. Development 102, 489–497. Baker, N.E. (2000). Notch signaling in the nervous system. Pieces still missing from the puzzle. BioEssays 22, 264–273. Baker, N.E., Mlodzik, M., and Rubin, G.M. (1990). Spacing differentiation in the developing Drosophila eye: a

180.

181.

182.

183.

184.

185.

186.

187. 188. 189.

190.

191.

192.

193.

194.

195.

196.

197.

ﬁbrinogen-related lateral inhibitor encoded by scabrous. Science 250, 1370–1377. Baker, N.E. and Rubin, G.M. (1989). Effect on eye development of dominant mutations in Drosophila homologue of the EGF receptor. Nature 340, 150–153. Baker, N.E. and Rubin, G.M. (1989). Ellipse mutations in the Drosophila homologue of the EGF receptor affect pattern formation, cell division, and cell death in eye imaginal discs. Dev. Biol. 150, 381–396. Baker, N.E., Yu, S., and Han, D. (1996). Evolution of proneural atonal expression during distinct regulatory phases in the developing Drosophila eye. Curr. Biol. 6, 1290–1301. Baker, N.E. and Yu, S.-Y. (1997). Proneural function of neurogenic genes in the developing Drosophila eye. Curr. Biol. 7, 122–132. [See also 2001 Development 128, 3889– 3898.] Baker, N.E. and Yu, S.-Y. (1998). The R8-photoreceptor equivalence group in Drosophila: fate choice precedes regulated Delta transcription and is independent of Notch gene dose. Mechs. Dev. 74, 3–14. Baker, N.E. and Yu, S.-Y. (2001). The EGF receptor deﬁnes domains of cell cycle progression and survival to regulate cell number in the developing Drosophila eye. Cell 104, 699–708. [See also 2001 Sems. Cell Dev. Biol. 12, 499–507.] Baker, N.E. and Zitron, A.E. (1995). Drosophila eye development: Notch and Delta amplify a neurogenic pattern conferred on the morphogenetic furrow by scabrous. Mechs. Dev. 49, 173–189. Baker, T.A. and Bell, S.P. (1998). Polymerases and the replisome: machines within machines. Cell 92, 295–305. Baker, W.K. (1967). A clonal system of differential gene activity in Drosophila. Dev. Biol. 16, 1–17. Baker, W.K. (1978). A clonal analysis reveals early developmental restrictions in the Drosophila head. Dev. Biol. 62, 447–463. Baker, W.K., Marcey, D.J., and McElwain, M.C. (1985). On the development of ectopic eyes in Drosophila melanogaster produced by the mutation extra eye (ee). Genetics 111, 67–88. Balcells, L., Modolell, J., and Ruiz-G´omez, M. (1988). A unitary basis for different Hairy-wing mutations of Drosophila melanogaster. EMBO J. 7, 3899–3906. Bally-Cuif, L., Dubois, L., and Vincent, A. (1998). Molecular cloning of Zcoe2, the zebraﬁsh homolog of Xenopus Xcoe2 and mouse EBF-2, and its expression during primary neurogenesis. Mechs. Dev. 77, 85–90. Bandziulis, R.J., Swanson, M.S., and Dreyfuss, G. (1989). RNA-binding proteins as developmental regulators. Genes Dev. 3, 431–437. Banerjee, U., Renfranz, P.J., Hinton, D.R., Rabin, B.A., and Benzer, S. (1987). The sevenless+ protein is expressed apically in cell membranes of developing Drosophila retina; it is not restricted to cell R7. Cell 51, 151–158. Banerjee, U., Renfranz, P.J., Pollock, J.A., and Benzer, S. (1987). Molecular characterization and expression of sevenless, a gene involved in neuronal pattern formation in the Drosophila eye. Cell 49, 281–291. Banerjee, U. and Zipursky, S.L. (1990). The role of cell-cell interaction in the development of the Drosophila visual system. Neuron 4, 177–187. Bang, A.G., Bailey, A.M., and Posakony, J.W. (1995). Hairless promotes stable commitment to the sensory organ precursor cell fate by negatively regulating the activity of the Notch signaling pathway. Dev. Biol. 172, 479–494.

REFERENCES

198. Bang, A.G., Hartenstein, V., and Posakony, J.W. (1991). Hairless is required for the development of adult sensory organ precursor cells in Drosophila. Development 111, 89– 104. 199. Bang, A.G. and Kintner, C. (2000). Rhomboid and Star facilitate presentation and processing of the Drosophila TGFα homolog Spitz. Genes Dev. 14, 177–186. [See also 2001 references: Cell 107, 161–171 and 173–182.] 200. Bang, A.G. and Posakony, J.W. (1992). The Drosophila gene Hairless encodes a novel basic protein that controls alternative cell fates in adult sensory organ development. Genes Dev. 6, 1752–1769. 201. Bangs, P. and White, K. (2000). Regulation and execution of apoptosis during Drosophila development. Dev. Dynamics 218, 68–79. 202. Bannister, A.J. and Kouzarides, T. (1996). The CBP coactivator is a histone acetyltransferase. Nature 384, 641– 643. 203. Bantignies, F., Goodman, R.H., and Smolik, S.M. (2000). Functional interaction between the coactivator Drosophila CREB-binding protein and ASH1, a member of the trithorax group of chromatin modiﬁers. Mol. Cell. Biol. 20, 9317–9330. [See also 2001 Science 294, 1331–1334.] 204. Baonza, A., Casci, T., and Freeman, M. (2001). A primary role for the epidermal growth factor receptor in ommatidial spacing in the Drosophila eye. Curr. Biol. 11, 396– 404. 205. Baonza, A., de Celis, J.F., and Garc´ıa-Bellido, A. (2000). Relationships between extramacrochaetae and Notch signalling in Drosophila wing development. Development 127, 2383–2393. 206. Baonza, A. and Garc´ıa-Bellido, A. (1999). Dual role of extramacrochaetae in cell proliferation and cell differentiation during wing morphogenesis in Drosophila. Mechs. Dev. 80, 133–146. 207. Baonza, A., Roch, F., and Mart´ın-Blanco, E. (2000). DER signaling restricts the boundaries of the wing ﬁeld during Drosophila development. Proc. Natl. Acad. Sci. USA 97, 7331–7335. 208. Bar-Sagi, D., Rotin, D., Batzer, A., Mandiyan, V., and Schlessinger, J. (1993). SH3 domains direct cellular localization of signaling molecules. Cell 74, 83–91. 209. Barbash, D.A. and Cline, T.W. (1995). Genetic and molecular analysis of the autosomal component of the primary sex determination signal of Drosophila melanogaster. Genetics 141, 1451–1471. 210. Bard, J.B.L. (1984). Butterﬂy wing patterns: how good a determining mechanism is the simple diffusion of a single morphogen? J. Embryol. Exp. Morph. 84, 255–274. 211. Bardwell, V.J. and Treisman, R. (1994). The POZ domain: A conserved protein-protein interaction motif. Genes Dev. 8, 1664–1677. ¨ 212. Barges, S., Mihaly, J., Galloni, M., Hagstrom, K., Muller, M., Shanower, G., Schedl, P., Gyurkovics, H., and Karch, F. (2000). The Fab-8 boundary deﬁnes the distal limit of the bithorax complex iab-7 domain and insulates iab-7 from initiation elements and a PRE in the adjacent iab-8 domain. Development 127, 779–790. 213. Barinaga, M. (1991). Dimers direct development. Science 251, 1176–1177. 214. Barinaga, M. (1995). Focusing on the eyeless gene. Science 267, 1766–1767. 215. Barinaga, M. (1995). Two major signaling pathways meet at MAP kinase. Science 269, 1673.

313

216. Barlow, P.W. (1978). Endopolyploidy: towards an understanding of its biological signiﬁcance. Acta Biotheor. 27, 1–18. 217. Barnes, J., ed. (1984). “The Complete Works of Aristotle.” Vol. 1. Princeton Univ. Pr., Princeton, N. J. [N.B.: Aristotle’s comment about vinegar “gnats” – which Peyer (1947) and Sturtevant (1965, p. 43) conclude must be D. melanogaster – is in History of Animals, Book 5, part 19, line 552b5; cf. Book 4:8:535a4, and Generation of Animals, Book 1:16:721a5 ff.] 218. Barolo, S., Walker, R.G., Polyanovsky, A.D., Freschi, G., Keil, T., and Posakony, J.W. (2000). A Notch-independent activity of Suppressor of Hairless is required for normal mechanoreceptor physiology. Cell 103, 957–969. [See also 2001 Curr. Biol. 11, 789–792.] 219. Barrington, E.J.W., ed. (1979). “Hormones and Evolution.” Vol. 1. Acad. Pr., New York. 220. Barrio, R., de Celis, J.F., Bolshakov, S., and Kafatos, F.C. (1999). Identiﬁcation of regulatory regions driving the expression of the Drosophila spalt complex at different developmental stages. Dev. Biol. 215, 33–47. 221. Barrio, R., Shea, M.J., Carulli, J., Lipkow, K., Gaul, U., Frommer, G., Schuh, R., J¨ackle, H., and Kafatos, F.C. (1996). The spalt-related gene of Drosophila melanogaster is a member of an ancient gene family, deﬁned by the adjacent, region-speciﬁc homeotic gene spalt. Dev. Genes Evol. 206, 315–325. 222. Bartel, P.L., Chien, C.-T., Sternglanz, R., and Fields, S. (1993). Using the two-hybrid system to detect proteinprotein interactions. In “Cellular Interactions in Development: A Practical Approach,” (D.A. Hartley, ed.). Oxford Univ. Pr.: New York, pp. 153–179. 223. Bashirullah, A., Halsell, S.R., Cooperstock, R.L., Kloc, M., Karaiskakis, A., Fisher, W.W., Fu, W., Hamilton, J.K., Etkin, L.D., and Lipshitz, H.D. (1999). Joint action of two RNA degradation pathways controls the timing of maternal transcript elimination at the midblastula transition in Drosophila melanogaster. EMBO J. 18, 2610–2620. 224. Basler, K. (2000). Waiting periods, instructive signals and positional information. EMBO J. 19, 1169–1175. 225. Basler, K., Christen, B., and Hafen, E. (1991). Ligandindependent activation of the Sevenless receptor tyrosine kinase changes the fate of cells in the developing Drosophila eye. Cell 64, 1069–1081. 226. Basler, K. and Hafen, E. (1988). Control of photoreceptor cell fate by the sevenless protein requires a functional tyrosine kinase domain. Cell 54, 299–311. 227. Basler, K. and Hafen, E. (1988). Sevenless and Drosophila eye development: a tyrosine kinase controls cell fate. Trends Genet. 4, 74–79. 228. Basler, K. and Hafen, E. (1989). Dynamics of Drosophila eye development and temporal requirements of sevenless expression. Development 107, 723–731. 229. Basler, K. and Hafen, E. (1991). Speciﬁcation of cell fate in the developing eye of Drosophila. BioEssays 13, 621–631. 230. Basler, K., Siegrist, P., and Hafen, E. (1989). The spatial and temporal expression pattern of sevenless is exclusively controlled by gene-internal elements. EMBO J. 8, 2381– 2386. 231. Basler, K. and Struhl, G. (1994). Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368, 208–214. 232. Basler, K., Yen, D., Tomlinson, A., and Hafen, E. (1990). Reprogramming cell fate in the developing Drosophila

314

233. 234.

235.

236.

237.

238.

239.

240.

241.

242.

243.

244. 245.

246.

247.

248.

REFERENCES

retina: transformation of R7 cells by ectopic expression of rough. Genes Dev. 4, 728–739. Bass, B.L. (2000). Double-stranded RNA as a template for gene silencing. Cell 101, 235–238. Bastiani, M.J., De Couet, H.G., Quinn, J.M.A., Karlstrom, R.O., Kotrla, K., Goodman, C.S., and Ball, E.E. (1992). Position-speciﬁc expression of the annulin protein during grasshopper embryogenesis. Dev. Biol. 154, 129–142. Batchelor, A.H., Piper, D.E., de la Brousse, F.C., McKnight, S.L., and Wolberger, C. (1998). The structure of GABPα/β: an ETS domain-ankyrin repeat heterodimer bound to DNA. Science 279, 1037–1041. Bate, M. (1993). The mesoderm and its derivatives. In “The Development of Drosophila melanogaster,” Vol. 2 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Lab. Pr.: Plainview, N. Y., pp. 1013–1090. Bate, M. and Martinez Arias, A. (1991). The embryonic origin of imaginal discs in Drosophila. Development 112, 755–761. Bate, M. and Martinez Arias, A., eds. (1993). “The Development of Drosophila melanogaster.” Cold Spring Harbor Lab. Pr., Plainview, N. Y. Bate, M., Rushton, E., and Currie, D.A. (1991). Cells with persistent twist expression are the embryonic precursors of adult muscles in Drosophila. Development 113, 79–89. Bateson, W. (1894). “Materials for the Study of Variation Treated with Especial Regard to Discontinuity in the Origin of Species.” MacMillan, London. (N.B.: Quotes were excerpted from pages 16, 68, and 85.) Batterham, P., Crew, J.R., Sokac, A.M., Andrews, J.R., Pasquini, G.M.F., Davies, A.G., Stocker, R.F., and Pollock, J.A. (1996). Genetic analysis of the lozenge gene complex in Drosophila melanogaster : adult visual system phenotypes. J. Neurogenetics 10, 193–220. Baumgartner, S. and Noll, M. (1991). Network of interactions among pair-rule genes regulating paired expression during primordial segmentation of Drosophila. Mechs. Dev. 33, 1–18. Bausenwein, B.S., Schmidt, M., Mielke, B., and Raabe, T. (2000). In vivo functional analysis of the Daughter of Sevenless protein in receptor tyrosine kinase signaling. Mechs. Dev. 90, 205–215. Bax, B. and Jhoti, H. (1995). Putting the pieces together. Curr. Biol. 5, 1119–1121. Baxevanis, A.D. and Vinson, C.R. (1993). Interactions of coiled coils in transcription factors: where is the speciﬁcity? Curr. Op. Gen. Dev. 3, 278–285. Bayer, C., von Kalm, L., and Fristrom, J.W. (1996). Gene regulation in imaginal disc and salivary gland development during Drosophila metamorphosis. In “Metamorphosis: Postembryonic Reprogramming of Gene Expression in Amphibian and Insect Cells,” (L.I. Gilbert, J.R. Tata, and B.G. Atkinson, eds.). Acad. Pr.: New York, pp. 321– 361. Bayer, C.A., Holley, B., and Fristrom, J.W. (1996). A switch in Broad-Complex zinc-ﬁnger isoform expression is regulated posttranscriptionally during the metamorphosis of Drosophila imaginal discs. Dev. Biol. 177, 1–14. [See also 2001 Development 128, 3729–3737.] Bayer, C.A., von Kalm, L., and Fristrom, J.W. (1997). Relationships between protein isoforms and genetic functions demonstrate functional redundancy at the BroadComplex during Drosophila metamorphosis. Dev. Biol. 187, 267–282.

249. Baylies, M.K. and Bate, M. (1996). twist : a myogenic switch in Drosophila. Science 272, 1481–1484. 250. Baylies, M.K., Bate, M., and Ruiz Gomez, M. (1997). The speciﬁcation of muscle in Drosophila. Cold Spr. Harb. Symp. Quant. Biol. 62, 385–393. 251. Baylies, M.K., Bate, M., and Ruiz Gomez, M. (1998). Myogenesis: a view from Drosophila. Cell 93, 921–927. 252. Bazett-Jones, D.P., Cˆote, J., Landel, C.C., Peterson, C.L., and Workman, J.L. (1999). The SWI/SNF complex creates loop domains in DNA and polynucleosome arrays and can disrupt DNA-histone contacts within these domains. Mol. Cell. Biol. 19, 1470–1478. 253. Bazett-Jones, D.P., Leblanc, B., Herfort, M., and Moss, T. (1994). Short-range DNA looping by the Xenopus HMG-box transcription factor, xUBF. Science 264, 1134– 1137. 254. Beachy, P.A. (1990). A molecular view of the Ultrabithorax homeotic gene of Drosophila. Trends Genet. 6, 46–51. 255. Beachy, P.A., Cooper, M.K., Young, K.E., von Kessler, D.P., Park, W.-J., Tanaka Hall, T.M., Leahy, D.J., and Porter, J.A. (1997). Multiple roles of cholesterol in Hedgehog protein biogenesis and signaling. Cold Spr. Harb. Symp. Quant. Biol. 62, 191–204. 256. Beachy, P.A., Helfand, S.L., and Hogness, D.S. (1985). Segmental distribution of bithorax complex proteins during Drosophila development. Nature 313, 545–551. 257. Beadle, G.W. (1970). Alfred Henry Sturtevant (1891–1970). Amer. Philos. Soc. Yearbook 1970, 166–171. 258. Beamonte, D. and Modolell, J. (1989). Search for Drosophila genes encoding a conserved domain present in the achaete-scute complex and myc proteins. Mol. Gen. Genet. 215, 281–285. 259. Beardsley, T. (1991). Smart genes. Sci. Am. 265 #2, 86–95. ¨ 260. Becker, H.J. (1957). Uber R¨ontgenmosaikﬂecken und Defektmutationen am Auge von Drosophila und die Entwicklungsphysiologie des Auges. Z. Indukt. Abstamm. Vererbungsl. 88, 333–373. 261. Becker, H.J. (1966). Genetic and variegation mosaics in the eye of Drosophila. Curr. Top. Dev. Biol. 1, 155–171. 262. Becskei, A. and Serrano, L. (2000). Engineering stability in gene networks by autoregulation. Nature 405, 590–593. 263. Beeman, R.W., Stuart, J.J., Brown, S.J., and Denell, R.E. (1993). Structure and function of the homeotic gene complex (HOM-C) in the beetle, Tribolium castaneum. BioEssays 15, 439–444. 264. Begemann, G., Michon, A.-M., van der Voorn, L., Wepf, R., and Mlodzik, M. (1995). The Drosophila orphan nuclear receptor Seven-up requires the Ras pathway for its function in photoreceptor determination. Development 121, 225–235. 265. Begemann, G., Paricio, N., Artero, R., Kiss, I., P´erez-Alonso, M., and Mlodzik, M. (1997). muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys3 His-type zinc-ﬁnger-containing proteins. Development 124, 4321–4331. ¨ 266. Behrens, J., von Kries, J.P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R., and Birchmeier, W. (1996). Functional interaction of β-catenin with the transcription factor LEF1. Nature 382, 638–642. 267. Beiman, M., Shilo, B.-Z., and Volk, T. (1996). Heartless, a Drosophila FGF receptor homolog, is essential for cell migration and establishment of several mesodermal lineages. Genes Dev. 10, 2993–3002. 268. Beitel, G.J. and Krasnow, M.A. (2000). Genetic control of

REFERENCES

269. 270. 271.

272.

273.

274.

275.

276.

277.

278.

279. 280.

281.

282. 283.

284.

285.

286.

287.

288.

epithelial tube size in the Drosophila tracheal system. Development 127, 3271–3282. Bejsovec, A. (1999). Signal transduction: Wnt signalling shows its versatility. Curr. Biol. 9, R684–R687. Bejsovec, A. (2000). Wnt signaling: an embarrassment of receptors. Curr. Biol. 10, R919–R922. Bejsovec, A. and Martinez Arias, A. (1991). Roles of wingless in patterning the larval epidermis of Drosophila. Development 113, 471–485. Bejsovec, A. and Wieschaus, E. (1993). Segment polarity gene interactions modulate epidermal patterning in Drosophila embryos. Development 119, 501–517. Bejsovec, A. and Wieschaus, E. (1995). Signaling activities of the Drosophila wingless gene are separately mutable and appear to be transduced at the cell surface. Genetics 139, 309–320. Bell, A.C. and Felsenfeld, G. (1999). Stopped at the border: boundaries and insulators. Curr. Opin. Gen. Dev. 9, 191– 198. Bell, A.C., West, A.G., and Felsenfeld, G. (2001). Insulators and boundaries: versatile regulatory elements in the eukaryotic genome. Science 291, 447–450. Bella¨ıche, Y., Gho, M., Kaltschmidt, J.A., Brand, A.H., and Schweisguth, F. (2001). Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division. Nature Cell Biol. 3, 50–57. Bellaiche, Y., The, I., and Perrimon, N. (1998). Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion. Nature 394, 85– 88. Bellen, H.J., O’Kane, C.J., Wilson, C., Grossniklaus, U., Pearson, R.K., and Gehring, W.J. (1989). P-elementmediated enhancer detection: a versatile method to study development in Drosophila. Genes Dev. 3, 1288–1300. Bellen, H.J. and Smith, R.F. (1995). FlyBase: a virtual Drosophila cornucopia. Trends Genet. 11, 456–457. Bellen, H.J., Wilson, C., and Gehring, W.J. (1990). Dissecting the complexity of the nervous system by enhancer detection. BioEssays 12, 199–204. Belote, J.M. and Baker, B.S. (1982). Sex determination in Drosophila melanogaster: analysis of transformer-2, a sextransforming locus. Proc. Natl. Acad. Sci. USA 79, 1568– 1572. Bender, W. (1985). Homeotic gene products as growth factors. Cell 43, 559–560. Bender, W., Akam, M., Karch, F., Beachy, P.A., Peifer, M., Spierer, P., Lewis, E.B., and Hogness, D.S. (1983). Molecular genetics of the bithorax complex in Drosophila melanogaster. Science 221, 23–29. Bender, W. and Hudson, A. (2000). P element homing to the Drosophila bithorax complex. Development 127, 3981–3992. Benedyk, M.J., Mullen, J.R., and DiNardo, S. (1994). oddpaired: a zinc ﬁnger pair-rule protein required for the timely activation of engrailed and wingless in Drosophila embryos. Genes Dev. 8, 105–117. Benezra, R. (1994). An intermolecular disulﬁde bond stabilizes E2A homodimers and is required for DNA binding at physiological temperatures. Cell 79, 1057–1067. Benlali, A., Draskovic, I., Hazelett, D.J., and Treisman, J.E. (2000). act up controls actin polymerization to alter cell shape and restrict Hedgehog signaling in the Drosophila eye disc. Cell 101, 271–281. Bennett, D.H. and Alphey, L.S. (2001). Role of PP1 in

315

289.

290.

291. 292. 293.

294.

295.

296.

297. 298.

299.

300.

301.

302.

303.

304.

305.

dpp/TGFβ signalling. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a139. Bennett, R.L. and Hoffmann, F.M. (1992). Increased levels of the Drosophila Abelson tyrosine kinase in nerves and muscles: subcellular localization and mutant phenotypes imply a role in cell-cell interactions. Development 116, 953–966. Benveniste, R.J., Thor, S., Thomas, J.B., and Taghert, P.H. (1998). Cell type-speciﬁc regulation of the Drosophila FMRF-NH2 neuropeptide gene by Apterous, a LIM homeodomain transcription factor. Development 125, 4757– 4765. Benzer, S. (1971). From the gene to behavior. JAMA (J. Amer. Med. Assoc.) 218, 1015–1022. Benzer, S. (1973). Genetic dissection of behavior. Sci. Am. 229 #6, 24–37. Benzer, S. (1991). The ﬂy and eye. In “Development of the Visual System,” Proc. Retina Res. Found. Symp., Vol. 3 (D.M.-K. Lam and C.J. Shatz, eds.). MIT Press: Cambridge, Mass., pp. 9–34. Berardi, M.J., Sun, C., Zehr, M., Abildgaard, F., Peng, J., Speck, N.A., and Bushweller, J.H. (1999). The Ig fold of the core binding factor a Runt domain is a member of a family of structurally and functionally related Ig-fold DNAbinding domains. Structure 7, 1247–1256. Berdnik, D., T¨or¨ok, T., Bulgheresi, S., Petronczki, M., and Knoblich, J.A. (2000). Genetic control of asymmetric cell division. Proc. 41st Ann. Drosophila Res. Conf. Abstracts Vol., a253. Berg, J.M. and Shi, Y. (1996). The galvanization of biology: a growing appreciation for the roles of zinc. Science 271, 1081–1085. Berkowitz, A. (1996). Networks of neurons, networks of genes. Neuron 17, 199–202. Bernard, F. (1937). Recherches sur la morphog´en`ese des yeux compos´es d’arthropodes. D´eveloppement, croissance, r´eduction. Bull. Biol. Fr. Belg. Suppl. 23, 1–162 (+ 6 plates). Bernﬁeld, M., G¨otte, M., Park, P.W., Reizes, O., Fitzgerald, M.L., Lincecum, J., and Zako, M. (2000). Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68, 729–777. Berry, M. and Gehring, W. (2000). Phosphorylation status of the SCR homeodomain determines its functional activity: essential role for protein phosphatase 2A,B . EMBO J. 19, 2946–2957. Bershadsky, A., Chausovsky, A., Becker, E., Lyubimova, A., and Geiger, B. (1996). Involvement of microtubules in the control of adhesion-dependent signal transduction. Curr. Biol. 6, 1279–1289. Berthelsen, J., Kilstrup-Nielsen, C., Blasi, F., Mavilio, F., and Zappavigna, V. (1999). The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH. Genes Dev. 13, 946–953. Bertuccioli, C., Fasano, L., Jun, S., Wang, S., Sheng, G., and Desplan, C. (1996). In vivo requirement for the paired domain and homeodomain of the paired segmentation gene product. Development 122, 2673–2685. Bessette, D.C. and Verheyen, E.M. (2001). Genetic characterization of nemo function during development in Drosophila melanogaster. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a205. Bettler, D., Pearson, S., and Yedvobnick, B. (1996). The

316

306.

307.

308.

309.

310.

311.

312.

313.

314.

315.

316.

317. 318.

319.

320.

321.

322.

REFERENCES

nuclear protein encoded by the Drosophila neurogenic gene mastermind is widely expressed and associates with speciﬁc chromosomal regions. Genetics 143, 859–875. ¨ Beuchle, D., Struhl, G., and Muller, J. (2001). Polycomb group proteins and heritable silencing of Drosophila Hox genes. Development 128, 993–1004. Bhadra, U., Pal-Bhadra, M., and Birchler, J.A. (1999). Role of the male speciﬁc lethal (msl) genes in modifying the effects of sex chromosomal dosage in Drosophila. Genetics 152, 249–268. Bhadra, U., Pal-Bhadra, M., and Birchler, J.A. (2001). Evidence that the MSL complex blocks a response of genes to histone acetylation. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a101. Bhalla, U.S. and Iyengar, R. (1999). Emergent properties of networks of biological signaling pathways. Science 283, 381–387. Bhanot, P., Brink, M., Samos, C.H., Hsieh, J.-C., Wang, Y., Macke, J.P., Andrew, D., Nathans, J., and Nusse, R. (1996). A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382, 225–230. Bhanot, P., Fish, M., Jemison, J.A., Nusse, R., Nathans, J., and Cadigan, K.M. (1999). Frizzled and DFrizzled2 function as redundant receptors for Wingless during Drosophila embryonic development. Development 126, 4175–4186. Bhat, K.M. (1998). frizzled and frizzled 2 play a partially redundant role in Wingless signaling and have similar requirements to Wingless in Neurogenesis. Cell 95, 1027– 1036. Bhat, K.M., Poole, S.J., and Schedl, P. (1995). The mitimere and pdm1 genes collaborate during speciﬁcation of the RP2/sib lineage in Drosophila neurogenesis. Mol. Cell. Biol. 15, 4052–4063. Bhat, K.M. and Schedl, P. (1994). The Drosophila miti-mere gene, a member of the POU family, is required for the speciﬁcation of the RP2/sibling lineage during neurogenesis. Development 120, 1483–1501. Bhojwani, J., Singh, A., Misquitta, L., Mishra, A., and Sinha, P. (1995). Search for Drosophila genes based on patterned expression of mini-white reporter gene of a P lacW vector in adult eyes. Roux’s Arch. Dev. Biol. 205, 114–121. Bianchi, M.E., Falciola, L., Ferrari, S., and Lilley, D.M.J. (1992). The DNA binding site of HMG1 protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins. EMBO J. 11, 1055–1063. Bianchi, M.E. and Lilley, D.M.J. (1995). Applying a genetic cantilever. Nature 375, 532. Biddle, R.L. (1932). The bristles of hybrids between Drosophila melanogaster and Drosophila simulans. Genetics 17, 153–174. Biehs, B., Franc¸ois, V., and Bier, E. (1996). The Drosophila short gastrulation gene prevents Dpp from autoactivating and suppressing neurogenesis in the neuroectoderm. Genes Dev. 10, 2922–2934. Biehs, B., Sturtevant, M.A., and Bier, E. (1998). Boundaries in the Drosophila wing imaginal disc organize veinspeciﬁc genetic programs. Development 125, 4245–4257. Bienz, M. (1997). Endoderm induction in Drosophila: the nuclear targets of the inducing signals. Curr. Opin. Gen. Dev. 7, 683–688. Bienz, M. (1999). APC: the plot thickens. Curr. Opin. Gen. Dev. 9, 595–603.

¨ 323. Bienz, M. and Muller, J. (1995). Transcriptional silencing of homeotic genes in Drosophila. BioEssays 17, 775–784. ¨ ¨ B., and 324. Bienz, M., Saari, G., Tremml, G., Muller, J., Zust, Lawrence, P.A. (1988). Differential regulation of Ultrabithorax in two germ layers of Drosophila. Cell 53, 567– 576. 325. Bienz, M. and Tremml, G. (1988). Domain of Ultrabithorax expression in Drosophila visceral mesoderm from autoregulation and exclusion. Nature 333, 576–578. 326. Bier, E. (1998). Localized activation of RTK/MAPK pathways during Drosophila development. BioEssays 20, 189– 194. 327. Bier, E. (2000). “The Coiled Spring: How Life Begins.” Cold Spr. Harb. Lab. Pr., Cold Spring Harbor, N. Y. 328. Bier, E. (2000). Drawing lines in the Drosophila wing: initiation of wing vein development. Curr. Opin. Gen. Dev. 10, 393–398. 329. Bier, E., H., V., Shepherd, S., Lee, K., McCall, K., Barbel, S., Ackerman, L., Carretto, R., Uemura, T., Grell, E., Jan, L.Y., and Jan, Y.N. (1989). Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector. Genes Dev. 3, 1273–1287. 330. Bier, E., Jan, L.Y., and Jan, Y.N. (1990). rhomboid, a gene required for dorsoventral axis establishment and peripheral nervous system development in Drosophila melanogaster. Genes Dev. 4, 190–203. 331. Bier, E., Vaessin, H., Younger-Shepherd, S., Jan, L.Y., and Jan, Y.N. (1992). deadpan, an essential pan-neural gene in Drosophila, encodes a helix-loop-helix protein similar to the hairy gene product. Genes Dev. 6, 2137–2151. 332. Biggs, W.H., III, Zavitz, K.H., Dickson, B., van der Straten, A., Brunner, D., Hafen, E., and Zipursky, S.L. (1994). The Drosophila rolled locus encodes a MAP kinase required in the sevenless signal transduction pathway. EMBO J. 13, 1628–1635. 333. Bilder, D., Li, M., and Perrimon, N. (2000). Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113–116. 334. Bilder, D. and Scott, M.P. (1998). Hedgehog and Wingless induce metameric pattern in the Drosophila visceral mesoderm. Dev. Biol. 201, 43–56. ¨ ¨ ¨ 335. Billeter, M., Guntert, P., Luginbuhl, P., and Wuthrich, K. (1996). Hydration and DNA recognition by homeodomains. Cell 85, 1057–1065. 336. Billin, A.N., Cockerill, K.A., and Poole, S.J. (1991). Isolation of a family of Drosophila POU domain genes expressed in early development. Mechs. Dev. 34, 75–84. 337. Billin, A.N. and Poole, S.J. (1995). Expression domains of the Cf1a POU domain protein during Drosophila development. Roux’s Arch. Dev. Biol. 204, 502–508. 338. Binari, R.C., Staveley, B.E., Johnson, W.A., Godavarti, R., Sasisekharan, R., and Manoukian, A.S. (1997). Genetic evidence that heparin-like glycosaminoglycans are involved in wingless signaling. Development 124, 2623–2632. 339. Bingham, P.M. (1997). Cosuppression comes to the animals. Cell 90, 385–387. 340. Birchler, J.A., Pal Bhadra, M., and Bhadra, U. (2000). Making noise about silence: repression of repeated genes in animals. Curr. Opin. Gen. Dev. 10, 211–216. 341. Birdsall, K., Zimmerman, E., Teeter, K., and Gibson, G. (2000). Genetic variation for the positioning of wing veins in Drosophila melanogaster. Evol. Dev. 2, 16–24. 342. Birge, R.B. and Hanafusa, H. (1993). Closing in on SH2 speciﬁcity. Science 262, 1522–1524.

REFERENCES

343. Birmingham, L. (1942). Boundaries of differentiation of cephalic imaginal discs in Drosophila. J. Exp. Zool. 91, 345– 363. 344. Bishop, S.A., Klein, T., Martinez Arias, A., and Couso, J.P. (1999). Composite signalling from Serrate and Delta establishes leg segments in Drosophila through Notch. Development 126, 2993–3003. 345. Black, D.L. (2000). Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology. Cell 103, 367–370. 346. Black, R.A. and White, J.M. (1998). ADAMs: focus on the protease domain. Curr. Opin. Cell Biol. 10, 654–659. 347. Blackman, R.K., Sanicola, M., Raftery, L.A., Gillevet, T., and Gelbart, W.M. (1991). An extensive 3 cis-regulatory region directs the imaginal disk expression of decapentaplegic, a member of the TGF-β family in Drosophila. Development 111, 657–665. 348. Blackwell, T.K. and Weintraub, H. (1990). Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection. Science 250, 1104–1110. 349. Blackwood, E.M. and Kadonaga, J.T. (1998). Going the distance: a current view of enhancer action. Science 281, 60–63. 350. Blair, S.S. (1992). engrailed expression in the anterior lineage compartment of the developing wing blade of Drosophila. Development 115, 21–33. 351. Blair, S.S. (1992). shaggy (zeste-white 3) and the formation of supernumerary bristle precursors in the developing wing blade of Drosophila. Dev. Biol. 152, 263–278. 352. Blair, S.S. (1993). Mechanisms of compartment formation: evidence that non-proliferating cells do not play a critical role in deﬁning the D/V lineage restriction in the developing wing of Drosophila. Development 119, 339–351. 353. Blair, S.S. (1994). A role for the segment polarity gene shaggy-zeste white 3 in the speciﬁcation of regional identity in the developing wing of Drosophila. Dev. Biol. 162, 229–244. 354. Blair, S.S. (1995). Compartments and appendage development in Drosophila. BioEssays 17, 299–309. 355. Blair, S.S. (1995). Hedgehog digs up an old friend. Nature 373, 656–657. 356. Blair, S.S. (1996). Notch and Wingless signals collide. Science 271, 1822–1823. 357. Blair, S.S. (1997). Limb development: Marginal Fringe beneﬁts. Curr. Biol. 7, R686–R690. 358. Blair, S.S. (1999). Drosophila imaginal disc development: patterning the adult ﬂy. In “Development: Genetics, Epigenetics and Environmental Regulation,” (V.E.A. Russo, D.J. Cove, L.G. Edgar, R. Jaenisch, and F. Salamini, eds.). Springer: N. Y., pp. 347–370. 359. Blair, S.S. (1999). Eye development: Notch lends a handedness. Curr. Biol. 9, R356–R360. 360. Blair, S.S. (2000). Notch signaling: Fringe really is a glycosyltransferase. Curr. Biol. 10, R608–R612. 361. Blair, S.S., Brower, D.L., Thomas, J.B., and Zavortink, M. (1994). The role of apterous in the control of dorsoventral compartmentalization and PS integrin gene expression in the developing wing of Drosophila. Development 120, 1805–1815. 362. Blair, S.S., Giangrande, A., Skeath, J.B., and Palka, J. (1992). The development of normal and ectopic sensilla in the wings of hairy and Hairy wing mutants of Drosophila. Mechs. Dev. 38, 3–16.

317

363. Blair, S.S. and Palka, J. (1985). Axon guidance in the wing of Drosophila. Trends Neurosci. 8, 284–288. 364. Blair, S.S. and Ralston, A. (1997). Smoothened-mediated Hedgehog signalling is required for the maintenance of the anterior-posterior lineage restriction in the developing wing of Drosophila. Development 124, 4053–4063. 365. Blanchet-Tournier, M.-F., Tricoire, H., Busson, D., and Lamour-Isnard, C. (1995). The segment-polarity gene fused is highly conserved in Drosophila. Gene 161, 157– 162. 366. Blank, V., Kourilsky, P., and Isra¨el, A. (1992). NF-κB and related proteins: Rel/dorsal homologies meet ankyrin-like repeats. Trends Biochem. Sci. 17, 135–140. 367. Blaumueller, C.M. and Mlodzik, M. (2000). The Drosophila tumor suppressor expanded regulates growth, apoptosis, and patterning during development. Mechs. Dev. 92, 251– 262. 368. Blaumueller, C.M., Qi, H., Zagouras, P., and ArtavanisTsakonas, S. (1997). Intracellular cleavage of Notch leads to a heterodimeric receptor on the plasma membrane. Cell 90, 281–291. 369. Blobel, C.P. (1997). Metalloprotease-disintegrins: links to cell adhesion and cleavage of TNFα and Notch. Cell 90, 589–592. 370. Blochlinger, K., Bodmer, R., Jack, J., Jan, L.Y., and Jan, Y.N. (1988). Primary structure and expression of a product from cut, a locus involved in specifying sensory organ identity in Drosophila. Nature 333, 629–635. 371. Blochlinger, K., Bodmer, R., Jan, L.Y., and Jan, Y.N. (1990). Patterns of expression of Cut, a protein required for external sensory organ development in wild-type and cut mutant Drosophila embryos. Genes Dev. 4, 1322–1331. 372. Blochlinger, K., Jan, L.Y., and Jan, Y.N. (1991). Transformation of sensory organ identity by ectopic expression of Cut in Drosophila. Genes Dev. 5, 1124–1135. 373. Blochlinger, K., Jan, L.Y., and Jan, Y.N. (1993). Postembryonic patterns of expression of cut, a locus regulating sensory organ identity in Drosophila. Development 117, 441– 450. 374. Bloomquist, B.T., Shortridge, R.D., Schneuwly, S., Perdew, M., Montell, C., Steller, H., Rubin, G., and Pak, W.L. (1988). Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction. Cell 54, 723–733. 375. Blumer, K.J. and Johnson, G.L. (1994). Diversity in function and regulation of MAP kinase pathways. Trends Biochem. Sci. 19, 236–240. 376. Bock, I.R. and Wheeler, M.R. (1972). The Drosophila melanogaster species group. In “Studies in Genetics,” Vol. 7 (Pub. #7213) (M.R. Wheeler, ed.). Univ. of Texas Pr.: Austin, Texas, pp. 1–102. 377. Bodenstein, D. (1950). The postembryonic development of Drosophila. In “Biology of Drosophila,” (M. Demerec, ed.). Hafner: New York, pp. 275–367. 378. Bodmer, R., Barbel, S., Sheperd, S., Jack, J.W., Jan, L.Y., and Jan, Y.N. (1987). Transformation of sensory organs by mutations of the cut locus of D. melanogaster. Cell 51, 293– 307. [See also 2001 Mechs. Dev. 105, 57–68.] 379. Bodmer, R., Jan, L.Y., and Jan, Y.-N. (1993). A late role for a subset of neurogenic genes to limit sensory precursor recruitments in Drosophila embryos. Roux’s Arch. Dev. Biol. 202, 371–381. 380. Bodnar, J.W. (1997). Programming the Drosophila embryo. J. Theor. Biol. 188, 391–445.

318

381. Boguski, M.S. and McCormick, F. (1993). Proteins regulating Ras and its relatives. Nature 366, 643–654. 382. Bohley, P. (1996). Surface hydrophobicity and intracellular degradation of proteins. Biol. Chem. 377, 425–435. 383. Bohmann, D., Ellis, M.C., Staszewski, L.M., and Mlodzik, M. (1994). Drosophila Jun mediates Ras-dependent photoreceptor determination. Cell 78, 973–986. 384. Bohn, H. (1965). Analyse der Regenerationf¨ahigkeit der Insektenextremit¨at durch Amputations- und Transplantationsversuche an Larven der Afrikanischen Schabe (Leucophaea maderae Fabr.). II. Achsendetermination. Roux’Arch. Entw.-mech. 156, 449–503. 385. Bohn, H. (1976). Regeneration of proximal tissues from a more distal amputation level in the insect leg (Blaberus craniifer, Blattaria). Dev. Biol. 53, 285–293. 386. Bohn, H. (1976). Tissue interactions in the regenerating cockroach leg. In “Insect Development,” Symp. Roy. Entomol. Soc. Lond., Vol. 8 (P.A. Lawrence, ed.). Wiley: New York, pp. 170–185. 387. Boissonade, J. (1994). Long-range inhibition. Nature 369, 188–189. 388. Boivin, A. and Dura, J.-M. (1998). In vivo chromatin accessibility correlates with gene silencing in Drosophila. Genetics 150, 1539–1549. 389. Bolinger, R.A. and Panganiban, G. (2001). Speciﬁcation of neural and imaginal identities in the embryonic limb primordium of Drosophila melanogaster. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a195. 390. Bonabeau, E. (1997). From classical models of morphogenesis to agent-based models of pattern formation. Artif. Life 3, 191–211. 391. Bonﬁni, L., Karlovich, C.A., Dasgupta, C., and Banerjee, U. (1992). The Son of sevenless gene product: a putative activator of Ras. Science 255, 603–606. 392. Bonﬁni, L., Migliaccio, E., Pelicci, G., Lanfrancone, L., and Pelicci, P. (1996). Not all Shc’s roads lead to Ras. Trends Biochem. Sci. 21, 257–261. 393. Bonifer, C. (2000). Developmental regulation of eukaryotic gene loci. Trends Genet. 16, 310–315. 394. Bonini, N.M., Bui, Q.T., Gray-Board, G.L., and Warrick, J.M. (1997). The Drosophila eyes absent gene directs ectopic eye formation in a pathway conserved between ﬂies and vertebrates. Development 124, 4819–4826. 395. Bonini, N.M. and Choi, K.-W. (1995). Early decisions in Drosophila eye morphogenesis. Curr. Opin. Gen. Dev. 5, 507–515. 396. Bonini, N.M. and Fortini, M.E. (1999). Surviving Drosophila eye development: integrating cell death with differentiation during formation of a neural structure. BioEssays 21, 991–1003. 397. Bonini, N.M., Leiserson, W.M., and Benzer, S. (1993). The eyes absent gene: genetic control of cell survival and differentiation in the developing Drosophila eye. Cell 72, 379– 395. 398. Bonini, N.M., Leiserson, W.M., and Benzer, S. (1998). Multiple roles of the eyes absent gene in Drosophila. Dev. Biol. 196, 42–57. 399. Boole, G. (1854).“An Investigation of the Laws of Thought, on which are founded the Mathematical Theories of Logic and Probabilities.” Walton and Maberley, London. 400. Bopp, D., Burri, M., Baumgartner, S., Frigerio, G., and Noll, M. (1986). Conservation of a large protein domain in the segmentation gene paired and in functionally related genes of Drosophila. Cell 47, 1033–1040.

REFERENCES

401. Bopp, D., Jamet, E., Baumgartner, S., Burri, M., and Noll, M. (1989). Isolation of two tissue-speciﬁc Drosophila paired box genes, Pox meso and Pox neuro. EMBO J. 8, 3447–3457. 402. Boriack-Sjodin, P.A., Margarit, S.M., Bar-Sagi, D., and Kuriyan, J. (1998). The structural basis of the activation of Ras by Sos. Nature 394, 337–343. 403. Bork, P. (1993). Hundreds of ankyrin-like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally? Proteins: Struct., Funct., Genet. 17, 363–374. 404. Bork, P., Downing, A.K., Kieffer, B., and Campbell, I.D. (1996). Structure and distribution of modules in extracellular proteins. Quart. Rev. Biophys. 29, 119–167. 405. Bornemann, D., Miller, E., and Simon, J. (1998). Expression and properties of wild-type and mutant forms of the Drosophila Sex comb on Midleg (SCM) repressor protein. Genetics 150, 675–686. 406. Borod, E.R. and Heberlein, U. (1998). Mutual regulation of decapentaplegic and hedgehog during the initiation of differentiation in the Drosophila retina. Dev. Biol. 197, 187– 197. 407. Botas, J. and Auwers, L. (1996). Chromosomal binding sites of Ultrabithorax homeotic proteins. Mechs. Dev. 56, 129– 138. 408. Botas, J., Moscoso del Prado, J., and Garc´ıa-Bellido, A. (1982). Gene-dose titration analysis in the search of transregulatory genes in Drosophila. EMBO J. 1, 307–310. 409. Botquin, V., Hess, H., Fuhrmann, G., Anastassiadis, C., Gross, M.K., Vriend, G., and Sch¨oler, H.R. (1998). New POU dimer conﬁguration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox2. Genes Dev. 12, 2073–2090. 410. Botstein, D. (1993). Genetics in the post-sequence era. Trends Genet. 9, 101. 411. Boube, M., Benassayag, C., Seroude, L., and Cribbs, D.L. (1997). Ras1-mediated modulation of Drosophila homeotic function in cell and segment identity. Genetics 146, 619–628. 412. Boube, M., Faucher, C., Joulia, L., Cribbs, D.L., and Bourbon, H.-M. (2000). Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity speciﬁcation. Genes Dev. 14, 2906–2917. 413. Boube, M., Seroude, L., and Cribbs, D.L. (1998). Homeotic proboscipedia cell identity functions respond to cell signaling pathways along the proximo-distal axis. Int. J. Dev. Biol. 42, 431–436. 414. Bouchard, M., St-Amand, J., and Cˆot´e, S. (2000). Combinatorial activity of pair-rule proteins on the Drosophila gooseberry early enhancer. Dev. Biol. 222, 135–146. 415. Boudreaux, H.B. (1979). Signiﬁcance of intersegmental tendon system in Arthropod phylogeny and a monophyletic classiﬁcation of Arthropoda. In “Arthropod Phylogeny,” (A.P. Gupta, ed.). Van Nostrand Reinhold: New York, pp. 551–586. 416. Boulet, A.M., Lloyd, A., and Sakonju, S. (1991). Molecular deﬁnition of the morphogenetic and regulatory functions and the cis-regulatory elements of the Drosophila Abd-B homeotic gene. Development 111, 393–405. 417. Boulianne, G.L., de la Concha, A., Campos-Ortega, J.A., Jan, L.Y., and Jan, Y.N. (1991). The Drosophila neurogenic gene neuralized encodes a novel protein and is expressed in precursors of larval and adult neurons. EMBO J. 10, 2975–2983. 418. Bourgouin, C., Lundgren, S.E., and Thomas, J.B. (1992).

REFERENCES

419.

420.

421.

422.

423.

424.

425.

426.

427.

428.

429.

430.

431.

432.

433.

434.

435.

apterous is a Drosophila LIM domain gene required for the development of a subset of embryonic muscles. Neuron 9, 549–561. Bourouis, M., Moore, P., Ruel, L., Grau, Y., Heitzler, P., and Simpson, P. (1990). An early embryonic product of the gene shaggy encodes a serine/threonine protein kinase related to the CDC28/cdc2+ subfamily. EMBO J. 9, 2877–2884. Boussy, I.A., Yu, H., Leone, M.A., Ogura, K., and Itoh, M. (2001). P element repression may involve “homologydependent gene silencing.” Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a38. Boutros, M., Mihaly, J., Bouwmeester, T., and Mlodzik, M. (2000). Signaling speciﬁcity by Frizzled receptors in Drosophila. Science 288, 1825–1828. [See also 2001 Development 128, 4829–4835.] Boutros, M. and Mlodzik, M. (1999). Dishevelled: at the crossroads of divergent intracellular signaling pathways. Mechs. Dev. 83, 27–37. Boutros, M., Paricio, N., Strutt, D.I., and Mlodzik, M. (1998). Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94, 109–118. Bowles, J., Schepers, G., and Koopman, P. (2000). Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Dev. Biol. 227, 239–255. Bownes, M. (1975). Adult deﬁciencies and duplications of head and thoracic structures resulting from microcautery of blastoderm stage Drosophila embryos. J. Embryol. Exp. Morph. 34, 33–54. Bownes, M., Bournias-Vardiabasis, N., and Spare, W.J. (1979). Genetic analysis of the spineless-aristapedia homoeotic mutants of Drosophila melanogaster. Mol. Gen. Genet. 174, 67–74. Bownes, M. and Seiler, M. (1977). Developmental effects of exposing Drosophila embryos to ether vapour. J. Exp. Zool. 199, 9–23. Bowtell, D.D.L., Simon, M.A., and Rubin, G.M. (1988). Nucleotide sequence and structure of the sevenless gene of Drosophila melanogaster. Genes Dev. 2, 620–634. Boyle, M., Bonini, N., and DiNardo, S. (1997). Expression and function of clift in the development of somatic gonadal precursors within the Drosophila mesoderm. Development 124, 971–982. Brabant, M.C. and Brower, D.L. (1993). PS2 integrin requirements in Drosophila embryo and wing morphogenesis. Dev. Biol. 157, 49–59. Brabant, M.C., Fristrom, D., Bunch, T.A., and Brower, D.L. (1996). Distinct spatial and temporal functions for PS integrins during Drosophila wing morphogenesis. Development 122, 3307–3317. Bradley, B.P. (1980). Developmental stability of Drosophila melanogaster under artiﬁcial and natural selection in constant and ﬂuctuating environments. Genetics 95, 1033– 1042. Braitenberg, V. (1967). Patterns of projection in the visual system of the ﬂy. I. Retina-lamina projections. Exp. Brain Res. 3, 271–298. Brakeﬁeld, P.M. and French, V. (1999). Butterﬂy wings: the evolution of development of colour patterns. BioEssays 21, 391–401. Brand, A.H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415.

319

436. Brand, M. and Campos-Ortega, J.A. (1988). Two groups of interrelated genes regulate early neurogenesis in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 197, 457– 470. 437. Brand, M. and Campos-Ortega, J.A. (1990). Second-site modiﬁers of the split mutation of Notch deﬁne genes involved in neurogenesis in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 198, 275–285. 438. Brand, M., Jarman, A.P., Jan, L.Y., and Jan, Y.N. (1993). asense is a Drosophila neural precursor gene and is capable of initiating sense organ formation. Development 119, 1–17. 439. Brasted, A. (1941). An analysis of the expression of the mutant “engrailed” in Drosophila melanogaster. Genetics 26, 347–373. 440. Bray, D. (1995). Protein molecules as computational elements in living cells. Nature 376, 307–312. 441. Bray, D. (1998). Signaling complexes: biophysical constraints on intracellular communication. Annu. Rev. Biophys. Biomol. Struct. 27, 59–75. 442. Bray, D. and Lay, S. (1997). Computer-based analysis of the binding steps in protein complex formation. Proc. Natl. Acad. Sci. USA 94, 13493–13498. 443. Bray, S. (1998). A Notch affair. Cell 93, 499–503. 444. Bray, S. (1998). Notch signalling in Drosophila: three ways to use a pathway. Sems. Cell Dev. Biol. 9, 591–597. 445. Bray, S. (1999). Drosophila development: Scalloped and Vestigial take wing. Curr. Biol. 9, R245–R247. 446. Bray, S. (2000). Notch. Curr. Biol. 10, R433–R435. 447. Bray, S. (2000). Planar polarity: out of joint? Curr. Biol. 10, R155–R158. 448. Bray, S. (2000). Speciﬁcity and promiscuity among proneural proteins. Neuron 25, 1–5. 449. Bray, S. and Furriols, M. (2001). Notch pathway: making sense of Suppressor of Hairless. Curr. Biol. 11, R217–R221. 450. Bredt, D.S. (2000). Reeling CASK into the nucleus. Nature 404, 241–242. 451. Breen, T.R. (1999). Mutant alleles of the Drosophila trithorax gene produce common and unusual homeotic and other developmental phenotypes. Genetics 152, 319–344. 452. Breen, T.R., Chinwalla, V., and Harte, P.J. (1995). Trithorax is required to maintain engrailed expression in a subset of engrailed-expressing cells. Mechs. Dev. 52, 89–98. 453. Breen, T.R. and Duncan, I.M. (1986). Maternal expression of genes that regulate the bithorax complex of Drosophila melanogaster. Dev. Biol. 118, 442–456. 454. Breen, T.R. and Harte, P.J. (1993). trithorax regulates multiple homeotic genes in the bithorax and Antennapedia complexes and exerts different tissue-speciﬁc, parasegment-speciﬁc and promoter-speciﬁc effects on each. Development 117, 119–134. 455. Breiling, A., Bonte, E., Ferrari, S., Becker, P.B., and Paro, R. (1999). The Drosophila Polycomb protein interacts with nucleosomal core particles in vitro via its repression domain. Mol. Cell. Biol. 19, 8451–8460. [See also 2001 Nature 412, 651–655.] 456. Breitling, R. and Gerber, J.-K. (2000). Origin of the paired domain. Dev. Genes Evol. 210, 644–650. 457. Brennan, C.A., Ashburner, M., and Moses, K. (1998). Ecdysone pathway is required for furrow progression in the developing Drosophila eye. Development 125, 2653– 2664. 458. Brennan, C.A., Li, T.-R., Bender, M., Hsiung, F., and Moses, K. (2001). Broad-complex, but not Ecdysone receptor, is

320

459.

460.

461.

462.

463. 464.

465. 466.

467.

468. 469.

470.

471.

472.

473. 474.

475.

476.

477.

478.

REFERENCES

required for progression of the morphogenetic furrow in the Drosophila eye. Development 128, 1–11. Brennan, K., Klein, T., Wilder, E., and Martinez Arias, A. (1999). Wingless modulates the effects of dominant negative Notch molecules in the developing wing of Drosophila. Dev. Biol. 216, 210–229. Brennan, K., Tateson, R., Lewis, K., and Martinez Arias, A. (1997). A functional analysis of Notch mutations in Drosophila. Genetics 147, 177–188. Brennan, K., Tateson, R., Lieber, T., Couso, J.P., Zecchini, V., and Martinez Arias, A. (1999). The Abruptex mutations of Notch disrupt the establishment of proneural clusters in Drosophila. Dev. Biol. 216, 230–242. Brenner, S. (1981). Genes and development. In “Cellular Controls in Differentiation,” (C.W. Lloyd and D.A. Rees, eds.). Acad. Pr.: New York, pp. 3–7. Brenner, S. (1993). Thoughts on genetics at the ﬁn de si`ecle. Trends Genet. 9, 104. Brenner, S., Dove, W., Herskowitz, I., and Thomas, R. (1990). Genes and development: molecular and logical themes. Genetics 126, 479–486. Brent, R. (2000). Genomic biology. Cell 100, 169–183. Brentrup, D., Lerch, H.-P., J¨ackle, H., and Noll, M. (2000). Regulation of Drosophila wing vein patterning: net encodes a bHLH protein repressing rhomboid and is repressed by rhomboid-dependent Egfr signalling. Development 127, 4729–4741. Brentrup, D., Lerch, H.-P., and Noll, M. (1996). The Drosophila net gene, required for intervein fate in wings, encodes a putative bHLH transcription factor. Experientia 52, A16. Bresnick, A.R. (1999). Molecular mechanisms of nonmuscle myosin-II regulation. Curr. Opin. Cell Biol. 11, 26–33. Bridges, C.B. (1920). The mutant crossveinless in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 6, 660– 663. Bridges, C.B. and Brehme, K.S. (1944). “The Mutants of Drosophila melanogaster.” Carnegie Inst. (Publication #552), Washington, D. C. Bridges, C.B. and Morgan, T.H. (1923). The thirdchromosome group of mutant characters of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 327, 1–251. Briscoe, J. and Ericson, J. (1999). The speciﬁcation of neuronal identity by graded sonic hedgehog signalling. Sems. Cell Dev. Biol. 10, 353–362. Britten, R.J. and Davidson, E.H. (1969). Gene regulation for higher cells: a theory. Science 165, 349–357. Britton, J.S. and Edgar, B.A. (1998). Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms. Development 125, 2149–2158. Brizuela, B.J. and Kennison, J.A. (1997). The Drosophila homeotic gene moira regulates expression of engrailed and HOM genes in imaginal tissues. Mechs. Dev. 65, 209– 220. Broadie, K.S. and Bate, M. (1991). The development of adult muscles in Drosophila: albation of identiﬁed muscle precursor cells. Development 113, 103–118. Broadus, J. and Doe, C.Q. (1997). Extrinsic cues, intrinsic cues and microﬁlaments regulate asymmetric protein localization in Drosophila neuroblasts. Curr. Biol. 7, 827– 835. Broadus, J., Fuerstenberg, S., and Doe, C.Q. (1998). Staufen-dependent localization of prospero mRNA con-

479.

480.

481.

482.

483.

484. 485.

486.

487.

488.

489.

490.

491.

492.

493.

494.

tributes to neuroblast daughter-cell fate. Nature 391, 792– 795. Broadus, J., McCabe, J.R., Endrizzi, B., Thummel, C.S., and Woodard, C.T. (1999). The Drosophila bFTZ-F1 orphan nuclear receptor provides competence for stage-speciﬁc responses to the steroid hormone ecdysone. Molec. Cell 3, 143–149. Brock, D.A. and Gomer, R.H. (1999). A cell-counting factor regulating structure size in Dictyostelium. Genes Dev. 13, 1960–1969. Brock, H.W. (1987). Sequence and genomic structure of ras homologues Dmras85D and Dmras64B of Drosophila melanogaster. Gene 51, 129–137. Brock, H.W. and van Lohuizen, M. (2001). The Polycomb group–no longer an exclusive club? Curr. Opin. Gen. Dev. 11, 175–181. Brodsky, M.H. and Steller, H. (1996). Positional information along the dorsal-ventral axis of the Drosophila eye: graded expression of the four-jointed gene. Dev. Biol. 173, 428–446. Brody, T. (1999). The Interactive Fly: gene networks, development and the Internet. Trends Genet. 15, 333–334. Brody, T. and Odenwald, W.F. (2000). Programmed transformations in neuroblast gene expression during Drosophila CNS lineage development. Dev. Biol. 226, 34–44. [See also 2001 references: Cell 106, 511–521; Dev. Cell 1, 313–314; Nature 413, 471–473.] Brogiolo, W., Stocker, H., Ikeya, T., Rintelen, F., Fernandez, R., and Hafen, E. (2001). An evolutionarily conserved function of the Drosophila insulin receptor and insulinlike peptides in growth control. Curr. Biol. 11, 213–221. Brook, W.J. and Cohen, S.M. (1996). Antagonistic interactions between Wingless and Decapentaplegic responsible for dorsal-ventral pattern in the Drosophila leg. Science 273, 1373–1377. Brook, W.J., Diaz-Benjumea, F.J., and Cohen, S.M. (1996). Organizing spatial pattern in limb development. Annu. Rev. Cell Dev. Biol. 12, 161–180. Brook, W.J., Ostaﬁchuk, L.M., Piorecky, J., Wilkinson, M.D., Hodgetts, D.J., and Russell, M.A. (1993). Gene expression during imaginal disc regeneration detected using enhancer-sensitive P-elements. Development 177, 1287– 1297. Brooke, N.M., Garcia-Fern`andez, J., and Holland, P.W.H. (1998). The ParaHox gene cluster is an evolutionary sister of the Hox gene cluster. Nature 392, 920–922. [See also 2001 J. Anat. 199, 13–23.] Brou, C., Logeat, F., Gupta, N., Bessia, C., LeBail, O., Doedens, J.R., Cumano, A., Roux, P., Black, R.A., and Isra¨el, A. (2000). A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Molec. Cell 5, 207–216. Brou, C., Logeat, F., Lecourtois, M., Vandekerckhove, J., Kourilsky, P., Schweisguth, F., and Isra¨el, A. (1994). Inhibition of the DNA-binding activity of Drosophila Suppressor of Hairless and of its human homolog, KBF2/RBP-Jκ, by direct protein-protein interaction with Drosophila Hairless. Genes Dev. 8, 2491–2503. [See also 2001 Curr. Biol. 11, 789–792.] Brower, D.L. (1984). Posterior-to-anterior transformation in engrailed wing imaginal disks of Drosophila. Nature 310, 496–497. Brower, D.L. (1985). The sequential compartmentalization of Drosophila segments revisited. Cell 41, 361–364.

REFERENCES

495. Brower, D.L. (1986). engrailed gene expression in Drosophila imaginal discs. EMBO J. 5, 2649–2656. 496. Brower, D.L. (1987). Ultrabithorax gene expression in Drosophila imaginal discs and larval nervous system. Development 101, 83–92. 497. Brower, D.L., Lawrence, P.A., and Wilcox, M. (1981). Clonal analysis of the undifferentiated wing disk of Drosophila. Dev. Biol. 86, 448–455. 498. Brower, D.L., Piovant, M., and Reger, L.A. (1985). Developmental analysis of Drosophila position-speciﬁc antigens. Dev. Biol. 108, 120–130. 499. Brower, D.L., Smith, R.J., and Wilcox, M. (1980). A monoclonal antibody speciﬁc for diploid epithelial cells in Drosophila. Nature 285, 403–405. 500. Brower, D.L., Smith, R.J., and Wilcox, M. (1982). Cell shapes on the surface of the Drosophila wing imaginal disc. J. Embryol. Exp. Morph. 67, 137–151. 501. Brown, C.E., Lechner, T., Howe, L., and Workman, J.L. (2000). The many HATs of transcriptional coactivators. Trends Biochem. Sci. 25, 15–19. 502. Brown, J.L., Sonoda, S., Ueda, H., Scott, M.P., and Wu, C. (1991). Repression of the Drosophila fushi tarazu (ftz) segmentation gene. EMBO J. 10, 665–674. 503. Brown, J.L. and Wu, C. (1993). Repression of Drosophila pair-rule segmentation genes by ectopic expression of tramtrack. Development 117, 45–58. 504. Brown, N.H., Gregory, S.L., and Martin-Bermudo, M.D. (2000). Integrins as mediators of morphogenesis in Drosophila. Dev. Biol. 223, 1–16. 505. Brown, N.H. and Hartley, D.A. (1994). Exploring signalling pathways. Nature 370, 414–415. 506. Brown, N.L., Paddock, S.W., Sattler, C.A., Cronmiller, C., Thomas, B.J., and Carroll, S.B. (1996). daughterless is required for Drosophila photoreceptor cell determination, eye morphogenesis, and cell cycle progression. Dev. Biol. 179, 65–78. 507. Brown, N.L., Sattler, C.A., Markey, D.R., and Carroll, S.B. (1991). hairy gene function in the Drosophila eye: normal expression is dispensable but ectopic expression alters cell fates. Development 113, 1245–1256. 508. Brown, N.L., Sattler, C.A., Paddock, S.W., and Carroll, S.B. (1995). Hairy and Emc negatively regulate morphogenetic furrow progression in the Drosophila eye. Cell 80, 879– 887. 509. Brown, S. and Hombr´ıa, J.C.-G. (2000). Drosophila grain encodes a GATA transcription factor required for cell rearrangement during morphogenesis. Development 127, 4867–4876. ¨ 510. Bruckner, K., Perez, L., Clausen, H., and Cohen, S. (2000). Glycosyltransferase activity of Fringe modulates NotchDelta interactions. Nature 406, 411–415. 511. Brummel, T., Abdollah, S., Haerry, T.E., Shimell, M.J., Merriam, J., Raftery, L., Wrana, J.L., and O’Connor, M.B. (1999). The Drosophila Activin receptor Baboon signals through dSmad2 and controls cell proliferation but not patterning during larval development. Genes Dev. 13, 98–111. 512. Brummel, T.J., Twombly, V., Marqu´es, G., Wrana, J.L., Newfeld, S.J., Attisano, L., Massagu´e, J., O’Connor, M.B., and Gelbart, W.M. (1994). Characterization and relationship of Dpp receptors encoded by the saxophone and thick veins genes in Drosophila. Cell 78, 251–261. 513. Brunk, B.P. and Adler, P.N. (1990). Aristapedioid: a gain of function, homeotic mutation in Drosophila melanogaster. Genetics 124, 145–156.

321

514. Brunk, B.P., Martin, E.C., and Adler, P.N. (1991). Molecular genetics of the Posterior Sex Combs/Suppressor 2 of zeste region of Drosophila: aberrant expression of the Suppressor 2 of zeste gene results in abnormal bristle development. Genetics 128, 119–132. 515. Brunner, A., Twardzik, T., and Schneuwly, S. (1994). The Drosophila giant lens gene plays a dual role in eye and optic lobe development: inhibition of differentiation of ommatidial cells and interference in photoreceptor axon guidance. Mechs. Dev. 48, 175–185. ¨ 516. Brunner, D., Ducker, K., Oellers, N., Hafen, E., Scholz, H., and Kl¨ambt, C. (1994). The ETS domain protein PointedP2 is a target of MAP kinase in the Sevenless signal transduction pathway. Nature 370, 386–389. 517. Brunner, D., Oellers, N., Szabad, J., Biggs, W.H., III, Zipursky, S.L., and Hafen, E. (1994). A gain-of-function mutation in Drosophila MAP kinase activates multiple receptor tyrosine kinase signaling pathways. Cell 76, 875– 888. 518. Brunner, E., Brunner, D., Fu, W., Hafen, E., and Basler, K. (1999). The dominant mutation Glazed is a gain-offunction allele of wingless that, similar to loss of APC, interferes with normal eye development. Dev. Biol. 206, 178–188. 519. Brunner, E., Peter, O., Schweizer, L., and Basler, K. (1997). pangolin encodes a Lef-1 homologue that acts downstream of Armadillo to transduce the Wingless signal in Drosophila. Nature 385, 829–833. 520. Bryant, P.J. (1969). Mosaicism. [Book review of “Genetic Mosaics and Other Essays” by C. Stern]. BioScience 19, 1126. 521. Bryant, P.J. (1970). Cell lineage relationships in the imaginal wing disc of Drosophila melanogaster. Dev. Biol. 22, 389–411. 522. Bryant, P.J. (1971). Regeneration and duplication following operations in situ on the imaginal discs of Drosophila melanogaster. Dev. Biol. 26, 637–651. 523. Bryant, P.J. (1974). Determination and pattern formation in the imaginal discs of Drosophila. Curr. Top. Dev. Biol. 8, 41–80. 524. Bryant, P.J. (1975). Pattern formation in the imaginal wing disc of Drosophila melanogaster : fate map, regeneration and duplication. J. Exp. Zool. 193, 49–78. 525. Bryant, P.J. (1975). Regeneration and duplication in imaginal discs. In “Cell Patterning,” (R. Porter and J. Rivers, eds.). Elsevier: Amsterdam, pp. 71–93. 526. Bryant, P.J. (1978). Pattern formation in imaginal discs. In “The Genetics and Biology of Drosophila,” Vol. 2c (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 229–335. 527. Bryant, P.J. (1979). Pattern formation, growth control and cell interactions in Drosophila imaginal discs. In “Determinants of Spatial Organization,” Symp. Soc. Dev. Biol., Vol. 37 (S. Subtelny and I.R. Konigsberg, eds.). Acad. Pr.: New York, pp. 295–316. 528. Bryant, P.J. (1987). Experimental and genetic analysis of growth and cell proliferation in Drosophila imaginal discs. In “Genetic Regulation of Development,” (W.F. Loomis, ed.). Alan R. Liss: New York, pp. 339–372. 529. Bryant, P.J. (1988). Localized cell death caused by mutations in a Drosophila gene coding for a transforming growth factor-β homolog. Dev. Biol. 128, 386–395. 530. Bryant, P.J. (1991). In memoriam: Howard A. Schneiderman (1927–1990). Dev. Biol. 146, 1–3.

322

531. Bryant, P.J. (1993). The Polar Coordinate Model goes molecular. Science 259, 471–472. 532. Bryant, P.J. (1996). Cell proliferation control in Drosophila: ﬂies are not worms. BioEssays 18, 781–784. 533. Bryant, P.J. (1997). Junction genetics. Dev. Genet. 20, 75–90. 534. Bryant, P.J. (1999). Fickle ﬁngers of cell fate? Curr. Biol. 9, R655–R657. 535. Bryant, P.J., Adler, P.N., Duranceau, C., Fain, M.J., Glenn, S., Hsei, B., James, A.A., Littleﬁeld, C.L., Reinhardt, C.A., Strub, S., and Schneiderman, H.A. (1978). Regulative interactions between cells from different imaginal disks of Drosophila melanogaster. Science 201, 928–930. 536. Bryant, P.J., Bryant, S.V., and French, V. (1977). Biological regeneration and pattern formation. Sci. Am. 237 #1, 66– 81. 537. Bryant, P.J. and Fraser, S.E. (1988). Wound healing, cell communication, and DNA synthesis during imaginal disc regeneration in Drosophila. Dev. Biol. 127, 197–208. 538. Bryant, P.J. and Girton, J.R. (1980). Genetics of pattern formation. In “Development and Neurobiology of Drosophila,” (O. Siddiqi, P. Babu, L.M. Hall, and J.C. Hall, eds.). Plenum: New York, pp. 109–127. 539. Bryant, P.J., Girton, J.R., and Martin, P. (1980). Physical and pattern continuity in the insect epidermis. In “Insect Biology in the Future,” (M. Locke and D.S. Smith, eds.). Acad. Pr.: New York, pp. 517–542. 540. Bryant, P.J. and Hsei, B.W. (1977). Pattern formation in asymmetrical and symmetrical imaginal discs of Drosophila melanogaster. Amer. Zool. 17, 595–611. 541. Bryant, P.J., Huettner, B., Held, L.I., Jr., Ryerse, J., and Szidonya, J. (1988). Mutations at the fat locus interfere with cell proliferation control and epithelial morphogenesis in Drosophila. Dev. Biol. 129, 541–554. 542. Bryant, P.J. and Levinson, P. (1985). Intrinsic growth control in the imaginal primordia of Drosophila, and the autonomous action of a lethal mutation causing overgrowth. Dev. Biol. 107, 355–363. 543. Bryant, P.J. and Schmidt, O. (1990). The genetic control of cell proliferation in Drosophila imaginal discs. In “Growth Factors in Cell and Developmental Biology,” J. Cell Sci. Suppl., Vol. 13 (M.D. Waterﬁeld, ed.). Company of Biologists, Ltd.: Cambridge, pp. 169–189. 544. Bryant, P.J. and Schneiderman, H.A. (1969). Cell lineage, growth, and determination in the imaginal leg discs of Drosophila melanogaster. Dev. Biol. 20, 263–290. 545. Bryant, P.J. and Schubiger, G. (1971). Giant and duplicated imaginal discs in a new lethal mutant of Drosophila melanogaster. Dev. Biol. 24, 233–263. 546. Bryant, P.J. and Simpson, P. (1984). Intrinsic and extrinsic control of growth in developing organs. Q. Rev. Biol. 59, 387–415. 547. Bryant, S.V., Bryant, P.J., and French, V. (1981). Distal regeneration and symmetry. Science 212, 993–1002. 548. Bryant, S.V. and Muneoka, K. (1986). Views of limb development and regeneration. Trends Genet. 2, 153–159. 549. Buchenau, P., Hodgson, J., Strutt, H., and Arndt-Jovin, D.J. (1998). The distribution of Polycomb-group proteins during cell division and development in Drosophila embryos: impact on models for silencing. J. Cell Biol. 141, 469–481. 550. Buckles, G.R., Smith, Z.D.J., and Katz, F.N. (1992). mip causes hyperinnervation of a retinotopic map in Drosophila by excessive recruitment of R7 photoreceptor cells. Neuron 8, 1015–1029. 551. Budd, G.E. (1999). Does evolution in body patterning

REFERENCES

552.

553.

554.

555.

556. 557.

558.

559.

560.

561. 562. 563.

564.

565.

566. 567.

568.

569.

570.

genes drive morphological change–or vice versa? BioEssays 21, 326–332. Buescher, M., Yeo, S.L., Udolph, G., Zavortink, M., Yang, X., Tear, G., and Chia, W. (1998). Binary sibling neuronal cell fate decisions in the Drosophila embryonic central nervous system are nonstochastic and require inscuteablemediated asymmetry of ganglion mother cells. Genes Dev. 12, 1858–1870. Bui, Q.T., Zimmerman, J.E., Liu, H., and Bonini, N.M. (2000). Molecular analysis of Drosophila eyes absent mutants reveals features of the conserved Eya domain. Genetics 155, 709–720. Bui, Q.T., Zimmerman, J.E., Liu, H., Gray-Board, G.L., and Bonini, N.M. (2000). Functional analysis of an eye enhancer of the Drosophila eyes absent gene: differential regulation by eye speciﬁcation genes. Dev. Biol. 221, 355–364. Bulinski, S.C., Qiu, X., Durtschi, R.B., and Zeng, C. (2001). Dissecting external sensory organ formation through gain-of-function genetics. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a268. Burak, W.R. and Shaw, A.S. (2000). Signal transduction: hanging on a scaffold. Curr. Opin. Cell Biol. 12, 211–216. Buratovich, M.A., Anderson, S., Gieseler, K., Pradel, J., and Wilder, E.L. (2000). DWnt-4 and Wingless have distinct activities in the Drosophila dorsal epidermis. Dev. Genes Evol. 210, 111–119. Buratovich, M.A. and Bryant, P.J. (1995). Duplication of l(2)gd imaginal discs in Drosophila is mediated by ectopic expression of wg and dpp. Dev. Biol. 168, 452–463. Buratovich, M.A. and Bryant, P.J. (1997). Enhancement of overgrowth by gene interactions in lethal(2)giant discs imaginal discs from Drosophila melanogaster. Genetics 147, 657–670. Buratovich, M.A., Phillips, R.G., and Whittle, J.R.S. (1997). Genetic relationships between the mutations spade and Sternopleural and the wingless gene in Drosophila development. Dev. Biol. 185, 244–260. Burbelo, P.D. and Hall, A. (1995). Hot numbers in signal transduction. Curr. Biol. 5, 95–96. Burden, S.J. (2000). Wnts as retrograde signals for axon and growth cone differentiation. Cell 100, 495–497. Burge, C.B., Tuschl, T., and Sharp, P.A. (1998). Splicing of precursors to mRNAs by the spliceosomes. In “The RNA World,” (R.F. Gesteland, T.R. Cech, and J.F. Atkins, eds.). Cold Spr. Harb. Lab. Pr.: Cold Spring Harbor, pp. 525–560. Burgess, E.A. and Duncan, I. (1990). Direct control of antennal identity by the spineless-aristapedia gene of Drosophila. Mol. Gen. Genet. 221, 347–352. Burgess, S.M. and Kleckner, N. (1999). Collisions between yeast chromosomal loci in vivo are governed by three layers of organization. Genes Dev. 13, 1871–1883. ¨ Burglin, T.R. (1991). The TEA domain: a novel, highly conserved DNA-binding motif. Cell 66, 11–12. ¨ Burglin, T.R. (1997). Analysis of TALE superclass homeobox genes (MEIS, PBC, KNOX, Iroquois, TGIF) reveals a novel domain conserved between plants and animals. Nucl. Acids Res. 25, 4173–4180. ¨ Burglin, T.R. (1998). The PBC domain contains a MEINOX domain: coevolution of Hox and TALE homeobox genes? Dev. Genes Evol. 208, 113–116. Burke, R. and Basler, K. (1996). Dpp receptors are autonomously required for cell proliferation in the entire developing Drosophila wing. Development 122, 2261–2269. Burke, R. and Basler, K. (1996). Hedgehog-dependent

REFERENCES

571.

572.

573.

574. 575.

576.

577. 578.

579. 580.

581. 582.

583.

584.

585.

586.

587.

patterning in the Drosophila eye can occur in the absence of Dpp signaling. Dev. Biol. 179, 360–368. Burke, R. and Basler, K. (1997). Hedgehog signaling in Drosophila eye and limb development–conserved machinery, divergent roles? Curr. Opin. Neurobiol. 7, 55–61. Burke, R., Nellen, D., Bellotto, M., Hafen, E., Senti, K.-A., Dickson, B.J., and Basler, K. (1999). Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modiﬁed Hedgehog from signaling cells. Cell 99, 803–815. [See also 2001 references: Curr. Biol. 11, 1147– 1152; Development 128, 5119–5127; Science 293, 2080– 2084.] Burris, P.A., Zhang, Y., Rusconi, J.C., and Corbin, V. (1998). The pore-forming and cytoplasmic domains of the neurogenic gene product, BIG BRAIN, are conserved between Drosophila virilis and Drosophila melanogaster. Gene 206, 69–76. Burrus, L.W. (1994). Wnt-1 as a short-range signaling molecule. BioEssays 16, 155–157. Burz, D.S., Rivera-Pomar, R., J¨ackle, H., and Hanes, S.D. (1998). Cooperative DNA-binding by Bicoid provides a mechanism for threshold-dependent gene activation in the Drosophila embryo. EMBO J. 17, 5998–6009. Bush, A., Hiromi, Y., and Cole, M. (1996). biparous: A novel bHLH gene expressed in neuronal and glial precursors in Drosophila. Dev. Biol. 180, 759–772 (cf. erratum: Dev. Biol. 205, 332). Bush, A., Hiromi, Y., and Cole, M. (1999). Erratum. Dev. Biol. 205, 332. Bush, G., diSibio, G., Miyamoto, A., Denault, J.-B., Leduc, R., and Weinmaster, G. (2001). Ligand-induced signaling in the absence of furin processing of Notch1. Dev. Biol. 229, 494–502. Bushweller, J.H. (2000). CBF–a biophysical perspective. Sems. Cell Dev. Biol. 11, 377–382. Busseau, I., Diederich, R.J., Xu, T., and Artavanis-Tsakonas, S. (1994). A member of the Notch group of interacting loci, deltex encodes a cytoplasmic basic protein. Genetics 136, 585–596. Busturia, A. and Lawrence, P.A. (1994). Regulation of cell number in Drosophila. Nature 370, 561–563. Busturia, A. and Morata, G. (1988). Ectopic expression of homeotic genes caused by the elimination of the Polycomb gene in Drosophila imaginal epidermis. Development 104, 713–720. Busturia, A., Vernos, I., Macias, A., Casanova, J., and Morata, G. (1990). Different forms of Ultrabithorax proteins generated by alternative splicing are functionally equivalent. EMBO J. 9, 3551–3555. Busturia, A., Wightman, C.D., and Sakonju, S. (1997). A silencer is required for maintenance of transcriptional repression throughout Drosophila development. Development 124, 4343–4350. [See also 2001 Genetics 158, 291– 307.] Butler, S.J., Ray, S., and Hiromi, Y. (1997). klingon, a novel member of the Drosophila immunoglobulin superfamily, is required for the development of the R7 photoreceptor neuron. Development 124, 781–792. Cabral, J.H.M., Petosa, C., Sutcliffe, M.J., Raza, S., Byron, O., Poy, F., Marfatia, S.M., Chishti, A.H., and Liddington, R.C. (1996). Crystal structure of a PDZ domain. Nature 382, 649–652. Cabrera, C.V. (1992). The generation of cell diversity during

323

588.

589.

590.

591.

592.

593.

594.

595.

596.

597.

598.

599.

600. 601.

602. 603.

604.

early neurogenesis in Drosophila. Development 115, 893– 901. Cabrera, C.V. and Alonso, M.C. (1991). Transcriptional activation by heterodimers of the achaete-scute and daughterless gene products of Drosophila. EMBO J. 10, 2965– 2973. Cabrera, C.V., Alonso, M.C., and Huikeshoven, H. (1994). Regulation of scute function by extramacrochaetae in vitro and in vivo. Development 120, 3595–3603. Cabrera, C.V., Alonso, M.C., Johnston, P., Phillips, R.G., and Lawrence, P.A. (1987). Phenocopies induced with antisense RNA identify the wingless gene. Cell 50, 659–663. Cabrera, C.V., Botas, J., and Garcia-Bellido, A. (1985). Distribution of Ultrabithorax proteins in mutants of Drosophila bithorax complex and its transregulatory genes. Nature 318, 569–571. Cacace, A.M., Michaud, N.R., Therrien, M., Mathes, K., Copeland, T., Rubin, G.M., and Morrison, D.K. (1999). Identiﬁcation of constitutive and Ras-inducible phosphorylation sites of KSR: implications for 14-3-3 binding, mitogen-activated protein kinase binding, and KSR overexpression. Mol. Cell. Biol. 19, 229–240. C´aceres, M., Barbadilla, A., and Ruiz, A. (1999). Recombination rate predicts inversion size in diptera. Genetics 153, 251–259. Cadavid, A.L.M., Ginzel, A., and Fischer, J.A. (2000). The function of the Drosophila Fat facets deubiquitinating enzyme in limiting photoreceptor cell number is intimately associated with endocytosis. Development 127, 1727–1736. Cadigan, K.M., Fish, M.P., Rulifson, E.J., and Nusse, R. (1998). Wingless repression of Drosophila frizzled 2 expression shapes the Wingless morphogen gradient in the wing. Cell 93, 767–777. Cadigan, K.M., Grossniklaus, U., and Gehring, W.J. (1994). Localized expression of sloppy paired protein maintains the polarity of Drosophila parasegments. Genes Dev. 8, 899–913. Cadigan, K.M. and Nusse, R. (1996). wingless signaling in the Drosophila eye and embryonic epidermis. Development 122, 2801–2812. Cadigan, K.M. and Nusse, R. (1997). Wnt signaling: a common theme in animal development. Genes Dev. 11, 3286– 3305. Cadigan, K.M., Parker, D.S., Jemison, J., Klinedienst, S., and Kohen, R. (2001). The role of the gammy legs gene in Wingless signaling. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a55. Cagan, R. (1993). Cell fate speciﬁcation in the developing Drosophila retina. Development 1993 Suppl., 19–28. Cagan, R.L., Kr¨amer, H., Hart, A.C., and Zipursky, S.L. (1992). The bride of sevenless and sevenless interaction: internalization of a transmembrane ligand. Cell 69, 393– 399. Cagan, R.L. and Ready, D.F. (1989). The emergence of order in the Drosophila pupal retina. Dev. Biol. 136, 346–362. Cagan, R.L. and Ready, D.F. (1989). Notch is required for successive cell decisions in the developing Drosophila retina. Genes Dev. 3, 1099–1112. Cagan, R.L., Thomas, B.J., and Zipursky, S.L. (1993). The role of induction in cell choice and cell cycle in the developing Drosophila retina. In “Molecular Basis of Morphogenesis,” Symp. Soc. Dev. Biol., Vol. 51 (M. Bernﬁeld, ed.). Wiley-Liss: New York, pp. 109–133.

324

605. Cagan, R.L. and Zipursky, S.L. (1992). Cell choice and patterning in the Drosophila retina. In “Determinants of Neuronal Identity,” (M. Shankland and E.R. Macagno, eds.). Acad. Pr.: New York, pp. 189–224. 606. Cai, H., N. and Shen, P. (2001). Effects of cis arrangement of chromatin insulators on enhancer-blocking activity. Science 291, 493–495. 607. Cai, H.N. and Levine, M. (1997). The gypsy insulator can function as a promoter-speciﬁc silencer in the Drosophila embryo. EMBO J. 16, 1732–1741. 608. Cai, J., Lan, Y., Appel, L.F., and Weir, M. (1994). Dissection of the Drosophila Paired protein: functional requirements for conserved motifs. Mechs. Dev. 47, 139–150. 609. Cai, Y., Chia, W., and Yang, X. (2001). A family of Snailrelated zinc ﬁnger proteins regulates two distinct and parallel mechanisms that mediate Drosophila neuroblast asymmetric divisions. EMBO J. 20, 1704–1714. 610. Cairns, J., Stent, G.S., and Watson, J.D., eds. (1966). “Phage and the Origins of Molecular Biology.” Cold Spring Harbor Lab. Quant. Biol., Cold Spring Harbor, N. Y. 611. Callaerts, P., Halder, G., and Gehring, W.J. (1997). Pax-6 in development and evolution. Annu. Rev. Neurosci. 20, 483–532. 612. Callahan, C.A., Bonkovsky, J.L., Scully, A.L., and Thomas, J.B. (1996). derailed is required for muscle attachment site selection in Drosophila. Development 122, 2761– 2767. 613. Callahan, P.S. (1979). Evolution of antennae, their sensilla and the mechanism of scent detection in Arthropoda. In “Arthropod Phylogeny,” (A.P. Gupta, ed.). Van Nostrand Reinhold: New York, pp. 259–298. 614. Calleja, M., Herranz, H., Estella, C., Casal, J., Lawrence, P., Simpson, P., and Morata, G. (2000). Generation of medial and lateral dorsal body domains by the pannier gene of Drosophila. Development 127, 3971–3980. 615. Calleja, M., Moreno, E., Pelaz, S., and Morata, G. (1996). Visualization of gene expression in living adult Drosophila. Science 274, 252–255. 616. Cambridge, S.B., Davis, R.L., and Minden, J.S. (1997). Drosophila mitotic domain boundaries as cell fate boundaries. Science 277, 825–828. 617. Campbell, G. and Tomlinson, A. (1995). Initiation of the proximodistal axis in insect legs. Development 121, 619– 628. 618. Campbell, G. and Tomlinson, A. (1998). The roles of the homeobox genes aristaless and Distal-less in patterning the legs and wings of Drosophila. Development 125, 4483– 4493. 619. Campbell, G. and Tomlinson, A. (1999). Transducing the Dpp morphogen gradient in the wing of Drosophila: regulation of Dpp targets by brinker. Cell 96, 553–562. 620. Campbell, G., Weaver, T., and Tomlinson, A. (1993). Axis speciﬁcation in the developing Drosophila appendage: the role of wingless, decapentaplegic, and the homeobox gene aristaless. Cell 74, 1113–1123. 621. Campbell, G.L. and Tomlinson, A. (2000). Transcriptional regulation of the Hedgehog effector CI by the zinc-ﬁnger gene combgap. Development 127, 4095–4103. 622. Campbell, J.H. (1985). An organizational interpretation of evolution. In “Evolution at a Crossroads: The New Biology and the New Philosophy of Science,” (D.J. Depew and B.H. Weber, eds.). MIT Pr.: Cambridge, Mass., pp. 133–167. 623. Campbell, S., Inamdar, M., Rodrigues, V., Raghavan, V., Palazzolo, M., and Chovnick, A. (1992). The scalloped gene encodes a novel, evolutionarily conserved transcrip-

REFERENCES

624.

625.

626.

627.

628.

629.

630.

631.

632.

633.

634.

635.

636.

637.

638.

639.

640.

tion factor required for sensory organ differentiation in Drosophila. Genes Dev. 6, 367–379. Campos, A.R., Fischbach, K.-F., and Steller, H. (1992). Survival of photoreceptor neurons in the compound eye of Drosophila depends on connections with the optic ganglia. Development 114, 355–366. Campos-Ortega, J.A. (1980). On compound eye development in Drosophila melanogaster. Curr. Top. Dev. Biol. 15, 347–371. Campos-Ortega, J.A. (1988). Cellular interactions during early neurogenesis of Drosophila melanogaster. Trends Neurosci. 11, 400–405. Campos-Ortega, J.A. (1993). Early neurogenesis in Drosophila melanogaster. In “The Development of Drosophila melanogaster,” Vol. 2 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Laboratory Pr.: New York, Campos-Ortega, J.A. (1998). The genetics of the Drosophila achaete-scute gene complex: a historical appraisal. Int. J. Dev. Biol. 42, 291–297. Campos-Ortega, J.A. and Gateff, E.A. (1976). The development of ommatidial patterning in metamorphosed eye imaginal discs implants of Drosophila melanogaster. W. Roux’s Arch. 179, 373–392. Campos-Ortega, J.A. and Haenlin, M. (1992). Regulatory signals and signal molecules in early neurogenesis of Drosophila melanogaster. Roux’s Arch. Dev. Biol. 201, 1–11. Campos-Ortega, J.A. and Hartenstein, V. (1997). “The Embryonic Development of Drosophila melanogaster.” 2nd ed. Springer, Berlin. ¨ Campos-Ortega, J.A., Jurgens, G., and Hofbauer, A. (1979). Cell clones and pattern formation: studies on sevenless, a mutant of Drosophila melanogaster. W. Roux’s Arch. 186, 27–50. Campos-Ortega, J.A. and Waitz, M. (1978). Cell clones and pattern formation: developmental restrictions in the compound eye of Drosophila. W. Roux’s Arch. 184, 155–170. Camps, M., Nichols, A., Gillieron, C., Antonsson, B., Muda, M., Chabert, C., Boschert, U., and Arkinstall, S. (1998). Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase. Science 280, 1262– 1265. Campuzano, S., Balcells, L., Villares, R., Carramolino, L., Garc´ıa-Alonso, L., and Modolell, J. (1986). Excess function of Hairy-wing mutations caused by gypsy and copia insertions within structural genes of the achaete-scute locus of Drosophila. Cell 44, 303–312. Campuzano, S., Carramolino, L., Cabrera, C.V., Ru´ızG´omez, M., Villares, R., Boronat, A., and Modolell, J. (1985). Molecular genetics of the achaete-scute gene complex of D. melanogaster. Cell 40, 327–338. Campuzano, S. and Modolell, J. (1992). Patterning of the Drosophila nervous system: the achaete-scute gene complex. Trends Genet. 8, 202–208. Canagarajah, B.J., Khokhlatchev, A., Cobb, M.H., and Goldsmith, E.J. (1997). Activation mechanism of the MAP kinase ERK2 by dual phosphorylation. Cell 90, 859– 869. Canon, J. and Banerjee, U. (2000). Runt and Lozenge function in Drosophila development. Sems. Cell Dev. Biol. 11, 327–336. Cant, K., Knowles, B.A., Mooseker, M.S., and Cooley, L. (1994). Drosophila Singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension. J. Cell Biol. 125, 369–380.

REFERENCES

641. Capdevila, J. and Belmonte, J.C.I. (1999). Extracellular modulation of the Hedgehog, Wnt and TGF-β signalling pathways during embryonic development. Curr. Opin. Gen. Dev. 9, 427–433. 642. Capdevila, J., Estrada, M.P., S´anchez-Herrero, E., and Guerrero, I. (1994). The Drosophila segment polarity gene patched interacts with decapentaplegic in wing development. EMBO J. 13, 71–82. 643. Capdevila, J. and Guerrero, I. (1994). Targeted expression of the signaling molecule decapentaplegic induces pattern duplications and growth alterations in Drosophila wings. EMBO J. 13, 4459–4468. 644. Capdevila, J., Pariente, F., Sampedro, J., Alonso, J.L., and Guerrero, I. (1994). Subcellular localization of the segment polarity protein patched suggests an interaction with the wingless reception complex in Drosophila embryos. Development 120, 987–998. 645. Capdevila, M.P., Botas, J., and Garc´ıa-Bellido, A. (1986). Genetic interactions between the Polycomb locus and the Antennapedia and Bithorax complexes of Drosophila. Roux’s Arch. Dev. Biol. 195, 417–432. 646. Capdevila, M.P. and Garcia-Bellido, A. (1974). Development and genetic analysis of bithorax phenocopies in Drosophila. Nature 250, 500–502. 647. Capdevila, M.P. and Garc´ıa-Bellido, A. (1978). Phenocopies of bithorax mutants. W. Roux’s Arch. 185, 105–126. 648. Capovilla, M. and Botas, J. (1998). Functional dominance among Hox genes: repression dominates activation in the regulation of dpp. Development 125, 4949–4957. 649. Carey, M. (1998). The enhanceosome and transcriptional synergy. Cell 92, 5–8. 650. Carlson, E.A. (1966). “The Gene: A Critical History.” W. B. Saunders, Philadephia. 651. Carlson, E.A. (1981). “Genes, Radiation, and Society: The Life and Work of H. J. Muller.” Cornell Univ. Pr., Ithaca, N. Y. 652. Carmena, A. and Baylies, M. (2001). Canoe: a possible link between Ras and Notch signaling pathways during Drosophila mesoderm differentiation. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a145. 653. Carmena, A., Murugasu-Oei, B., Menon, D., Jim´enez, F., and Chia, W. (1998). inscuteable and numb mediate asymmetric muscle progenitor cell divisions during Drosophila myogenesis. Genes Dev. 12, 304–315. 654. Carpenter, G. (2000). The EGF receptor: a nexus for trafﬁcking and signaling. BioEssays 22, 697–707. 655. Carr, A. and Biggin, M.D. (1999). A comparison of in vivo and in vitro DNA-binding speciﬁcities suggests a new model for homeoprotein DNA binding in Drosophila embryos. EMBO J. 18, 1598–1608. 656. Carramolino, L., Ruiz-Gomez, M., del Carmen Guerrero, M., Campuzano, S., and Modolell, J. (1982). DNA map of mutations at the scute locus of Drosophila melanogaster. EMBO J. 1, 1185–1191. 657. Carroll, S.B. (1990). Zebra patterns in ﬂy embryos: activation of stripes or repression of interstripes? Cell 60, 9–16. 658. Carroll, S.B. (1995). Homeotic genes and the evolution of arthropods and chordates. Nature 376, 479–485. 659. Carroll, S.B. (1998). From pattern to gene, from gene to pattern. Int. J. Dev. Biol. 42, 305–309. 660. Carroll, S.B. (2000). Endless forms: the evolution of gene regulation and morphological diversity. Cell 101, 577– 580. 661. Carroll, S.B., DiNardo, S., O’Farrell, P.H., White, R.A.H., and Scott, M.P. (1988). Temporal and spatial relationships

325

662.

663.

664.

665.

666. 667.

668.

669.

670.

671.

672.

673.

674.

675.

676.

677.

678.

679.

between segmentation and homeotic gene expression in Drosophila embryos: distributions of the fushi tarazu, engrailed, Sex combs reduced, Antennapedia, and Ultrabithorax proteins. Genes Dev. 2, 350–360. Carroll, S.B., Grenier, J.K., and Weatherbee, S.D. (2001). “From DNA to Diversity: Molecular Genetics and the Evolution of Animal Design.” Blackwell Science, Malden, Mass. Carroll, S.B., Laughon, A., and Thalley, B.S. (1988). Expression, function, and regulation of the hairy segmentation protein in the Drosophila embryo. Genes Dev. 2, 883–890. Carroll, S.B., Weatherbee, S.D., and Langeland, J.A. (1995). Homeotic genes and the regulation and evolution of insect wing number. Nature 375, 58–61. Carroll, S.B. and Whyte, J.S. (1989). The role of the hairy gene during Drosophila morphogenesis: stripes in imaginal discs. Genes Dev. 3, 905–916. Carson, H.L. (1986). Patterns of inheritance. Amer. Zool. 26, 797–809. Carson, H.L. and Bryant, P.J. (1979). Change in a secondary sexual character as evidence of incipient speciation in Drosophila silvestris. Proc. Natl. Acad. Sci. USA 76, 1929– 1932. Carson, H.L., Hardy, D.E., Spieth, H.T., and Stone, W.S. (1970). The evolutionary biology of the Hawaiian Drosophilidae. In “Essays in Evolution and Genetics,” (M.K. Hecht and W.C. Steere, eds.). Appleton-CenturyCrofts: New York, pp. 437–543. Carson, H.L. and Kaneshiro, K.Y. (1976). Drosophila of Hawaii: systematics and ecological genetics. Annu. Rev. Ecol. Syst. 7, 311–345. Carthew, R.W., Kauffmann, R.C., Kladny, S., Li, S., and Zhang, J. (1999). Cell determination in the Drosophila eye. In “Cell Lineage and Fate Determination,” (S.A. Moody, ed.). Acad. Pr.: New York, pp. 235–248. Carthew, R.W. and Rubin, G.M. (1990). seven in absentia, a gene required for speciﬁcation of R7 cell fate in the Drosophila eye. Cell 63, 561–577. Casanova, J. (1989). Mutations in the spalt gene of Drosophila cause ectopic expression of Ultrabithorax and Sex combs reduced. Roux’s Arch. Dev. Biol. 198, 137–140. Casanova, J., S´anchez-Herrero, E., and Morata, G. (1985). Contrabithorax and the control of spatial expression of the bithorax complex genes of Drosophila. J. Embryol. Exp. Morph. 90, 179–196. Casanova, J. and Struhl, G. (1993). The torso receptor localizes as well as transduces the spatial signal specifying terminal body pattern in Drosophila. Nature 362, 152–155. Casares, F., Bender, W., Merriam, J., and S´anchez-Herrero, E. (1997). Interactions of Drosophila Ultrabithorax regulatory regions with native and foreign promoters. Genetics 145, 123–137. Casares, F., Calleja, M., and S´anchez-Herrero, E. (1996). Functional similarity in appendage speciﬁcation by the Ultrabithorax and abdominal-A Drosophila HOX genes. EMBO J. 15, 3934–3942. Casares, F. and Mann, R.S. (1998). Control of antennal versus leg development in Drosophila. Nature 392, 723–726. [See also 2001 references: Curr. Biol. 11, R1025–R1027; Science 293, 1477–1480.] Casares, F. and Mann, R.S. (2000). A dual role for homothorax in inhibiting wing blade development and specifying proximal wing identities in Drosophila. Development 127, 1499–1508. Casares, F., S´anchez, L., Guerrero, I., and S´anchez-Herrero,

326

680.

681. 682.

683.

684.

685.

686.

687.

688.

689.

690.

691.

692.

693.

694.

695.

696.

697.

REFERENCES

E. (1997). The genital disc of Drosophila melanogaster. I. Segmental and compartmental organization. Dev. Genes Evol. 207, 216–228. Casares, F. and S´anchez-Herrero, E. (1995). Regulation of the infraabdominal regions of the bithorax complex of Drosophila by gap genes. Development 121, 1855–1866. Casci, T., Vin´os, J., and Freeman, M. (1999). Sprouty, an intracellular inhibitor of Ras signaling. Cell 96, 655–665. Castelli-Gair, J. (1998). Implications of the spatial and temporal regulation of Hox genes on development and evolution. Int. J. Dev. Biol. 42, 437–444. Castelli-Gair, J. (1998). The lines gene of Drosophila is required for speciﬁc functions of the Abdominal-B HOX protein. Development 125, 1269–1274. Castelli-Gair, J. and Akam, M. (1995). How the Hox gene Ultrabithorax speciﬁes two different segments: the signiﬁcance of spatial and temporal regulation within metameres. Development 121, 2973–2982. Castelli-Gair, J., Greig, S., Micklem, G., and Akam, M. (1994). Dissecting the temporal requirements for homeotic gene function. Development 120, 1983–1995. ¨ Castelli-Gair, J., Muller, J., and Bienz, M. (1992). Function of an Ultrabithorax minigene in imaginal cells. Development 114, 877–886. Cate, J.H., Yusupov, M.M., Yusupova, G.Z., Earnest, T.N., and Noller, H.F. (1999). X-ray crystal structures of 70S ribosome functional complexes. Science 285, 2095–2104. Caudy, M., Grell, E.H., Dambly-Chaudi`ere, C., Ghysen, A., Jan, L.Y., and Jan, Y.N. (1988). The maternal sex determination gene daughterless has zygotic activity necessary for the formation of peripheral neurons in Drosophila. Genes Dev. 2, 843–852. Caudy, M., V¨assin, H., Brand, M., Tuma, R., Jan, L.Y., and Jan, Y.N. (1988). daughterless, a Drosophila gene essential for both neurogenesis and sex determination, has sequence similarities to myc and the achaete-scute complex. Cell 55, 1061–1067. Cavalli, G. and Paro, R. (1999). Epigenetic inheritance of active chromatin after removal of the main transactivator. Science 286, 955–958. Cavallo, R., Rubenstein, D., and Peifer, M. (1997). Armadillo and dTCF: a marriage made in the nucleus. Curr. Opin. Gen. Dev. 7, 459–466. Cavallo, R.A., Cox, R.T., Moline, M.M., Roose, J., Polevoy, G.A., Clevers, H., Peifer, M., and Bejsovec, A. (1998). Drosophila Tcf and Groucho interact to repress Wingless signalling activity. Nature 395, 604–608. Cavener, D.R. (1987). Combinatorial control of structural genes in Drosophila: solutions that work for the animal. BioEssays 7, 103–107. Cavener, D.R. (1992). Transgenic animal studies on the evolution of genetic regulatory circuitries. BioEssays 14, 237–244. Caveney, S. (1985). Intercellular communication. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology,” Vol. 2 (G.A. Kerkut and L.I. Gilbert, eds.). Pergamon Pr.: Oxford, pp. 319–370. [See also 2001 Nature 411, 759–762.] Cavodeassi, F., del Corral, R.D., Campuzano, S., and Dom´ınguez, M. (1999). Compartments and organising boundaries in the Drosophila eye: the role of the homeodomain Iroquois proteins. Development 126, 4933–4942. Cavodeassi, F., Modolell, J., and Campuzano, S. (2000). The Iroquois homeobox genes function as dorsal selectors in the Drosophila head. Development 127, 1921–1929.

698. Celniker, S.E. (2000). The Drosophila genome. Curr. Opin. Gen. Dev. 10, 612–616. 699. Celniker, S.E., Sharma, S., Keelan, D.J., and Lewis, E.B. (1990). The molecular genetics of the bithorax complex of Drosophila: cis-regulation in the Abdominal-B domain. EMBO J. 9, 4277–4286. 700. Celotto, A.M. and Graveley, B.R. (2001). Alternative splicing of the Drosophila Dscam pre-mRNA is both temporally and spatially regulated. Genetics 159, 599–608. 701. Cerd´a-Olmedo, E. (1998). The development of concepts on development: a dialogue with Antonio Garc´ıa-Bellido. Int. J. Dev. Biol. 42, 233–236. 702. Ceresa, B.P. and Schmid, S.L. (2000). Regulation of signal transduction by endocytosis. Curr. Opin. Cell Biol. 12, 204– 210. 703. Certel, K., Hudson, A., Carroll, S.B., and Johnson, W.A. (2000). Restricted patterning of vestigial expression in Drosophila wing imaginal discs requires synergistic activation by both Mad and the Drifter POU domain transcription factor. Development 127, 3173–3183. 704. Chae, J., Kim, M.-J., Goo, J.H., Collier, S., Gubb, D., Charlton, J., Adler, P.N., and Park, W.J. (1999). The Drosophila tissue polarity gene starry night encodes a member of the protocadherin family. Development 126, 5421–5429. 705. Chakravarti, D., LaMorte, V.J., Nelson, M.C., Nakajima, T., Schulman, I.G., Juguilon, H., Montminy, M., and Evans, R.M. (1996). Role of CBP/P300 in nuclear receptor signalling. Nature 383, 99–103. 706. Chan, S.-K., Jaffe, L., Capovilla, M., Botas, J., and Mann, R.S. (1994). The DNA binding speciﬁcity of Ultrabithorax is modulated by cooperative interactions with Extradenticle, another homeoprotein. Cell 78, 603–615. 707. Chan, S.-K. and Mann, R.S. (1993). The segment identity functions of Ultrabithorax are contained within its homeo domain and carboxy-terminal sequences. Genes Dev. 7, 796–811. 708. Chan, S.-K., P¨opperl, H., Krumlauf, R., and Mann, R.S. (1996). An extradenticle-induced conformational change in a HOX protein overcomes an inhibitory function of the conserved hexapeptide motif. EMBO J. 15, 2476– 2487. 709. Chan, S.-K., Ryoo, H.-D., Gould, A., Krumlauf, R., and Mann, R.S. (1997). Switching the in vivo speciﬁcity of a minimal Hox-responsive element. Development 124, 2007–2014. 710. Chan, S.-K. and Struhl, G. (1997). Sequence-speciﬁc RNA binding by Bicoid. Nature 388, 634. 711. Chan, Y.-M. and Jan, Y.N. (1998). Roles for proteolysis and trafﬁcking in Notch maturation and signal transduction. Cell 94, 423–426. 712. Chan, Y.-M. and Jan, Y.N. (1999). Presenilins, processing of β-amyloid precursor protein, and Notch signaling. Neuron 23, 201–204. 713. Chandebois, R. (1977). Cell sociology and the problem of position effect: pattern formation, origin and role of gradients. Acta Biotheor. 26, 203–238. 714. Chandebois, R. and Faber, J. (1983). “Automation in Animal Development.” Monographs in Developmental Biology, Vol. 16. Karger, Basel. 715. Chang, H.-Y. and Ready, D.F. (2000). Rescue of photoreceptor degeneration in rhodopsin-null Drosophila mutants by activated Rac1. Science 290, 1978–1980. 716. Chang, H.C. and Rubin, G.M. (1997). 14-3-3 positively regulates Ras-mediated signaling in Drosophila. Genes Dev. 11, 1132–1139.

REFERENCES

717. Chang, H.C., Solomon, N.M., Wassarman, D.A., Karim, F.D., Therrien, M., Rubin, G.M., and Wolff, T. (1995). phyllopod functions in the fate determination of a subset of photoreceptors in Drosophila. Cell 80, 463– 472. 718. Chang, W.S., Zachow, K.R., and Bentley, D. (1993). Expression of epithelial alkaline phosphatase in segmentally iterated bands during grasshopper limb morphogenesis. Development 118, 651–663. 719. Chang, Y.-L., King, B.O., O’Connor, M., Mazo, A., and Huang, D.-H. (1995). Functional reconstruction of trans regulation of the Ultrabithorax promoter by the products of two antagonistic genes, trithorax and Polycomb. Mol. Cell. Biol. 15, 6601–6612. 720. Chang, Y.W. and Chien, C.T. (2001). Eye formation genes eyeless and dachshund are negatively regulated by proneural genes atonal and daughterless during Drosophila eye development. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a255. 721. Chang, Z., Price, B.D., Bockheim, S., Boedigheimer, M.J., Smith, R., and Laughon, A. (1993). Molecular and genetic characterization of the Drosophila tartan gene. Dev. Biol. 160, 315–332. 722. Chanut, F. and Heberlein, U. (1995). Role of the morphogenetic furrow in establishing polarity in the Drosophila eye. Development 121, 4085–4094. 723. Chanut, F. and Heberlein, U. (1997). Retinal morphogenesis in Drosophila: hints from an eye-speciﬁc decapentaplegic allele. Dev. Genet. 20, 197–207. 724. Chanut, F. and Heberlein, U. (1997). Role of decapentaplegic in initiation and progression of the morphogenetic furrow in the developing Drosophila retina. Development 124, 559–567. 725. Chanut, F., Luk, A., and Heberlein, U. (2000). A screen for dominant modiﬁers of roDom, a mutation that disrupts morphogenetic furrow progression in Drosophila, identiﬁes Groucho and Hairless as regulators of atonal expression. Genetics 156, 1203–1217. 726. Chao, J.L. and Sun, Y.H. (2001). eyg is a downstream effector of Notch signaling on cell proliferation in Drosophila eye. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a195. 727. Chaudhary, S., Brou, C., Valentin, M.-E., Burton, N., Tora, L., Chambon, P., and Davidson, I. (1994). A cell-speciﬁc factor represses stimulation of transcription in vitro by Transcriptional Enhancer Factor 1. Mol. Cell. Biol. 14, 5290–5299. 728. Chaudhary, S., Tora, L., and Davidson, I. (1995). Characterization of a HeLa cell factor which negatively regulates transcriptional activation in vitro by Transcriptional Enhancer Factor-1 (TEF-1). J. Biol. Chem. 270 #8, 3631–3637. 729. Cheah, P.Y., Chia, W., and Yang, X. (2000). Jumeaux, a novel Drosophila winged-helix family protein, is required for generating asymmetric sibling neuronal cell fates. Development 127, 3325–3335. 730. Chelsky, D., Ralph, R., and Jonak, G. (1989). Sequence requirements for synthetic peptide-mediated translocation to the nucleus. Mol. Cell. Biol. 9, 2487–2492. 731. Chen, C.-H., von Kessler, D.P., Park, W., Wang, B., Ma, Y., and Beachy, P.A. (1999). Nuclear trafﬁcking of Cubitus interruptus in the transcriptional regulation of Hedgehog target gene expression. Cell 98, 305–316. ¨ 732. Chen, C.-K., Kuhnlein, R.P., Eulenberg, K.G., Vincent, S., Affolter, M., and Schuh, R. (1998). The transcription factors KNIRPS and KNIRPS RELATED control cell migration

327

733.

734.

735.

736.

737.

738.

739.

740.

741.

742.

743.

744.

745.

746.

747.

748.

and branch morphogenesis during Drosophila tracheal development. Development 125, 4959–4968. Chen, C.-M., Freedman, J.A., Bettler, D.R., Jr., Manning, S.D., Giep, S.N., Steiner, J., and Ellis, H.M. (1996). polychaetoid is required to restrict segregation of sensory organ precursors from proneural clusters in Drosophila. Mechs. Dev. 57, 215–227. Chen, C.-m. and Struhl, G. (1999). Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila. Development 126, 5441–5452. Chen, E.H. and Baker, B.S. (1997). Compartmental organization of the Drosophila genital imaginal discs. Development 124, 205–218. Chen, F. and Rebay, I. (2000). split ends, a new component of the Drosophila EGF receptor pathway, regulates development of midline glial cells. Curr. Biol. 10, 943– 946. Chen, G., Fernandez, J., Mische, S., and Courey, A.J. (1999). A functional interaction between the histone deacetylase Rpd3 and the corepressor Groucho in Drosophila development. Genes Dev. 13, 2218–2230. Chen, G., Nguyen, P.H., and Courey, A.J. (1998). A role for Groucho tetramerization in transcriptional repression. Mol. Cell. Biol. 18, 7259–7268. Chen, H., Fre, S., Slepnev, V.I., Capua, M.R., Takei, K., Butler, M.H., Di Fiore, P.P., and De Camilli, P. (1998). Epsin is an EH-domain-binding protein implicated in clathrinmediated endocytosis. Nature 394, 793–797. Chen, M.S., Obar, R.A., Schroeder, C.C., Austin, T.W., Poodry, C.A., Wadsworth, S.C., and Vallee, R.B. (1991). Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis. Nature 351, 583–586. Chen, P., Nordstrom, W., Gish, B., and Abrams, J.M. (1996). grim, a novel cell death gene in Drosophila. Genes Dev. 10, 1773–1782. Chen, P., Rodriguez, A., Erskine, R., Thach, T., and Abrams, J.M. (1998). Dredd, a novel effector of the apoptosis activators reaper, grim, and hid in Drosophila. Dev. Biol. 201, 202–216. Chen, R., Amoui, M., Zhang, Z., and Mardon, G. (1997). Dachshund and Eyes absent proteins form a complex and function synergistically to induce ectopic eye development in Drosophila. Cell 91, 893–903. Chen, R., Halder, G., Zhang, Z., and Mardon, G. (1999). Signaling by the TGF-β homolog decapentaplegic functions reiteratively within the network of genes controlling retinal cell fate determination in Drosophila. Development 126, 935–943. Chen, T., Bunting, M., Karim, F.D., and Thummel, C.S. (1992). Isolation and characterization of ﬁve Drosophila genes that encode an ets-related DNA binding domain. Dev. Biol. 151, 176–191. Chen, X. and Fischer, J.A. (2000). In vivo structure/ function analysis of the Drosophila fat facets deubiquitinating enzyme gene. Genetics 156, 1829–1836. Chen, X., Li, Q., and Fischer, J.A. (2000). Genetic analysis of the Drosophila DNAprim gene: the function of the 60kD primase subunit of DNA polymerase opposes the fat facets signaling pathway in the developing eye. Genetics 156, 1787–1795. Chen, X., Overstreet, E., Wood, S.A., and Fischer, J.A. (2000). On the conservation of function of the Drosophila Fat facets deubiquitinating enzyme and Fam, its mouse homolog. Dev. Genes Evol. 210, 603–610.

328

749. Chen, Y., Cardinaux, J.-R., Goodman, R.H., and Smolik, S.M. (1999). Mutants of cubitus interruptus that are independent of PKA regulation are independent of hedgehog signaling. Development 126, 3607–3616. 750. Chen, Y., Fischer, W.H., and Gill, G.N. (1997). Regulation of the ERBB-2 promoter by RBPJκ and Notch. J. Biol. Chem. 272 #22, 14110–14114. 751. Chen, Y., Gallaher, N., Goodman, R.H., and Smolik, S.M. (1998). Protein kinase A directly regulates the activity and proteolysis of cubitus interruptus. Proc. Natl. Acad. Sci. USA 95, 2349–2354. [See also 2001 Genetics 158, 1157– 1166.] 752. Chen, Y., Goodman, R.H., and Smolik, S.M. (2000). Cubitus interruptus requires Drosophila CREB-binding protein to activate wingless expression in the Drosophila embryo. Mol. Cell. Biol. 20, 1616–1625. 753. Chen, Y., Riese, M.J., Killinger, M.A., and Hoffmann, F.M. (1998). A genetic screen for modiﬁers of Drosophila decapentaplegic signaling identiﬁes mutations in punt, Mothers against dpp and the BMP-7 homologue, 60 A. Development 125, 1759–1768. 754. Chen, Y. and Struhl, G. (1996). Dual roles for Patched in sequestering and transducing Hedgehog. Cell 87, 553–563. 755. Chen, Y. and Struhl, G. (1998). In vivo evidence that Patched and Smoothened constitute distinct binding and transducing components of a Hedgehog receptor complex. Development 125, 4943–4948. 756. Chen, Y.-G., Hata, A., Lo, R.S., Wotton, D., Shi, Y., Pavletich, N., and Massagu´e, J. (1998). Determinants of speciﬁcity in TGF-β signal transduction. Genes Dev. 12, 2144–2152. 757. Cheng, M.K. and Shearn, A. (2001). Molecular and genetic interactions between ash2, a trithorax group gene, and skittles, a gene encoding 1-phosphatidylinositol-4phosphate kinase. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a94. 758. Chern, J.J. and Choi, K.W. (2001). Cloning and characterizing of Lobe mutation. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a185. 759. Cheyette, B.N.R., Green, P.J., Martin, K., Garren, H., Hartenstein, V., and Zipursky, S.L. (1994). The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron 12, 977–996. 760. Chiang, C. and Beachy, P.A. (1994). Expression of a novel Toll-like gene spans the parasegment boundary and contributes to hedgehog function in the adult eye of Drosophila. Mechs. Dev. 47, 225–239. 761. Chicurel, M.E., Chen, C.S., and Ingber, D.E. (1998). Cellular control lies in the balance of forces. Curr. Opin. Cell Biol. 10, 232–239. 762. Chicurel, M.E., Singer, R.H., Meyer, C.J., and Ingber, D.E. (1998). Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions. Nature 392, 730–733. 763. Chien, C.-T., Bartel, P.L., Sternglanz, R., and Fields, S. (1991). The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA 88, 9578–9582. 764. Chien, C.-T., Hsiao, C.-D., Jan, L.Y., and Jan, Y.N. (1996). Neuronal type information encoded in the basic-helixloop-helix domain of proneural genes. Proc. Natl. Acad. Sci. USA 93, 13239–13244. 765. Chien, C.-T., Wang, S., Rothenberg, M., Jan, L.Y., and Jan, Y.N. (1998). Numb-associated kinase interacts with the

REFERENCES

766.

767.

768.

769.

770.

771.

772.

773.

774.

775.

776.

777.

778.

779.

780.

781.

782.

phosphotyrosine binding domain of Numb and antagonizes the function of Numb in vivo. Mol. Cell. Biol. 18, 598–607. Chihara, T. and Hayashi, S. (2000). Control of tracheal tubulogenesis by Wingless signaling. Development 127, 4433–4442. Child, G. (1935). Phenogenetic studies on scute-1 of Drosophila melanogaster. I. The associations between the bristles and the effects of genetic modiﬁers and temperature. Genetics 20, 109–126. Child, G. (1935). Phenogenetic studies on scute-1 of Drosophila melanogaster. II. The temperature-effective period. Genetics 20, 127–155. Child, G. (1936). Phenogenetic studies in scute of D. melanogaster. III. The effect of temperature on scute 5. Genetics 21, 808–816. Child, G.P., Blanc, R., and Plough, H.H. (1940). Somatic effects of temperature on development in Drosophila melanogaster. I. Phenocopies and reversal of dominance. Physiol. Zool. 13, 56–64. Childs, S.R. and O’Connor, M.B. (1994). Two domains of the tolloid protein contribute to its unusual genetic interaction with decapentaplegic. Dev. Biol. 162, 209–220. Childs, S.R., Wrana, J.L., Arora, K., Attisano, L., O’Connor, M.B., and Massagu´e, J. (1993). Identiﬁcation of a Drosophila activin receptor. Proc. Natl. Acad. Sci. USA 90, 9475–9479. Cho, K.-O., Chern, J., Izaddoost, S., and Choi, K.-W. (2000). Novel signaling from the peripodial membrane is essential for eye disc patterning in Drosophila. Cell 103, 331–342. Cho, K.-O. and Choi, K.-W. (1998). Fringe is essential for mirror symmetry and morphogenesis in the Drosophila eye. Nature 396, 272–276. Cho, K.W.Y. and Blitz, I.L. (1998). BMPs, Smads and metalloproteases: extracellular and intracellular modes of negative regulation. Curr. Opin. Gen. Dev. 8, 443–449. Choi, C.Y., Lee, Y.M., Kim, Y.H., Park, T., Jeon, B.H., Schulz, R.A., and Kim, Y. (1999). The homeodomain transcription factor NK-4 acts as either a transcriptional activator or repressor and interacts with the p300 coactivator and the Groucho corepressor. J. Biol. Chem. 274 #44, 31543–31552. Choi, K.-W. and Benzer, S. (1994). Rotation of photoreceptor clusters in the developing Drosophila eye requires the nemo gene. Cell 78, 125–136. Choi, K.-W., Mozer, B., and Benzer, S. (1996). Independent determination of symmetry and polarity in the Drosophila eye. Proc. Natl. Acad. Sci. USA 93, 5737–5741. Chou, W.-H., Hall, K.J., Wilson, D.B., Wideman, C.L., Townson, S.M., Chadwell, L.V., and Britt, S.G. (1996). Identiﬁcation of a novel Drosophila opsin reveals speciﬁc patterning of the R7 and R8 photoreceptor cells. Neuron 17, 1101–1115. Chou, W.-H., Huber, A., Bentrop, J., Schulz, S., Schwab, K., Chadwell, L.V., Paulsen, R., and Britt, S.G. (1999). Patterning of the R7 and R8 photoreceptor cells of Drosophila: evidence for induced and default cell-fate speciﬁcation. Development 126, 607–616. Choy, E., Chiu, V.K., Silletti, J., Feoktistov, M., Morimoto, T., Michaelson, D., Ivanov, I.E., and Philips, M.R. (1999). Endomembrane trafﬁcking of Ras: the CAAX motif targets proteins to the ER and Golgi. Cell 98, 69–80. Christen, B. and Bienz, M. (1994). Imaginal disc silencers from Ultrabithorax: evidence for Polycomb response elements. Mechs. Dev. 48, 255–266.

REFERENCES

783. Christian, J.L. (2000). BMP, Wnt and Hedgehog signals: how far can they go? Curr. Opin. Cell Biol. 12, 244–249. 784. Chu, J. and Panganiban, G. (2001). Distal-less speciﬁes antennal identity by interacting with Extradenticle and Homothorax. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a118. 785. Chu-LaGraff, Q., Wright, D.M., McNeil, L.K., and Doe, C.Q. (1991). The prospero gene encodes a divergent homeodomain protein that controls neuronal identity in Drosophila. Development Suppl. 2, 79–85. 786. Chuang, P.-T. and Kornberg, T.B. (2000). On the range of Hedgehog signaling. Curr. Opin. Gen. Dev. 10, 515–522. 787. Chun, J. (1999). Developmental neurobiology: a genetic Cheshire cat? Curr. Biol. 9, R651–R654. 788. Chung, C.-N., Hamaguchi, Y., Honjo, T., and Kawaichi, M. (1994). Site-directed mutagenesis study on DNA binding regions of the mouse homologue of Suppressor of Hairless, RBP-Jκ. Nucleic Acids Res. 22, 2938–2944. 789. Chung, Y.D., Zhu, J., Han, Y.-G., and Kernan, M.J. (2001). nompA encodes a PNS-speciﬁc, ZP domain protein required to connect mechanosensory dendrites to sensory structures. Neuron 29, 415–428. 790. Chuong, C.-M., Chodankar, R., Widelitz, R.B., and Jiang, T.-X. (2000). Evo-Devo of feathers and scales: building complex epithelial appendages. Curr. Opin. Gen. Dev. 10, 449–456. 791. Ciechanover, A., Orian, A., and Schwartz, A.L. (2000). Ubiquitin-mediated proteolysis: biological regulation via destruction. BioEssays 22, 442–451. 792. Ciechanska, E. and Brook, W. (2001). The role of l(3)1215 in joint formation in the Drosophila leg. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a196. 793. Cifuentes, F.J. and Garc´ıa-Bellido, A. (1997). Proximodistal speciﬁcation in the wing disc of Drosophila by the nubbin gene. Proc. Natl. Acad. Sci. USA 94, 11405– 11410. 794. Citri, Y., Colot, H.V., Jacquier, A.C., Yu, Q., Hall, J.C., Baltimore, D., and Rosbash, M. (1987). A family of unusually spliced biologically active transcripts encoded by a Drosophila clock gene. Nature 326, 42–47. 795. Clandinin, T.R. and Zipursky, S.L. (2000). Afferent growth cone interactions control synaptic speciﬁcity in the Drosophila visual system. Neuron 28, 427–436. 796. Clark, H.F., Brentrup, D., Schneitz, K., Bieber, A., Goodman, C., and Noll, M. (1995). Dachsous encodes a member of the cadherin superfamily that controls imaginal disc morphogenesis in Drosophila. Genes Dev. 9, 1530– 1542. 797. Clark, W.C. and Russell, M.A. (1977). The correlation of lysosomal activity and adult phenotype in a cell-lethal mutant of Drosophila. Dev. Biol. 57, 160–173. 798. Clarke, P.G.H. (1981). Chance, repetition, and error in the development of normal nervous systems. Persp. Biol. Med. 25, 2–19. 799. Clarke, P.R. (1994). Switching off MAP kinases. Curr. Biol. 4, 647–650. 800. Claxton, J.H. (1964). The determination of patterns with special reference to that of the central primary skin follicles in sheep. J. Theor. Biol. 7, 302–317. 801. Claxton, J.H. (1967). Patterns of abdominal tergite bristles in wild-type and scute Drosophila melanogaster. Genetics 55, 525–545. 802. Claxton, J.H. (1969). Mosaic analysis of bristle displacement in Drosophila. Genetics 63, 883–896.

329

803. Claxton, J.H. (1971). Cell numbers and acrostichal row numbers in hairy and non-hairy ﬂies. Dros. Info. Serv. 46, 133–134. 804. Claxton, J.H. (1974). Some quantitative features of Drosophila sternite bristle patterns. Aust. J. Biol. Sci. 27, 533–543. 805. Claxton, J.H. (1976). Developmental origin of even spacing between the microchaetes of Drosophila melanogaster. Aust. J. Biol. Sci. 29, 131–135. 806. Claxton, J.H. (1982). Temporal pattern of bristle development on Drosophila melanogaster sternites. Aust. J. Biol. Sci. 35, 653–660. 807. Claxton, J.H. and Kongsuwan, K. (1976). Non-autonomy in achaete mosaics of Drosophila. Genet. Res., Camb. 27, 11–22. 808. Cleghon, V., Feldmann, P., Ghiglione, C., Copeland, T.D., Perrimon, N., Hughes, D.A., and Morrison, D.K. (1998). Opposing actions of CSW and RasGAP modulate the strength of Torso RTK signaling in the Drosophila terminal pathway. Molec. Cell 2, 719–727. 809. Cleghon, V., Gayko, U., Copeland, T.D., Perkins, L.A., Perrimon, N., and Morrison, D.K. (1996). Drosophila terminal structure development is regulated by the compensatory activities of positive and negative phosphotyrosine signaling sites on the Torso RTK. Genes Dev. 10, 566–577. 810. Clevers, H. and van de Wetering, M. (1997). TCF/LEF factors earn their wings. Trends Genet. 13, 485–489. 811. Cleves, A.E. (1997). Protein transport: the nonclassical ins and outs. Curr. Biol. 7, R318–R320. ¨ 812. Clifford, R. and Schupbach, T. (1992). The torpedo (DER) receptor tyrosine kinase is required at multiple times during Drosophila embryogenesis. Development 115, 853–872. ¨ 813. Clifford, R. and Schupbach, T. (1994). Molecular analysis of the Drosophila EGF receptor homolog reveals that several genetically deﬁned classes of alleles cluster in subdomains of the receptor protein. Genetics 137, 531–550. ¨ 814. Clifford, R.J. and Schupbach, T. (1989). Coordinately and differentially mutable activities of torpedo, the Drosophila melanogaster homolog of the vertebrate EGF receptor gene. Genetics 123, 771–787. 815. Cline, T.W. (1989). The affairs of daughterless and the promiscuity of developmental regulators. Cell 59, 231– 234. 816. Cline, T.W. (1993). The Drosophila sex determination signal: how do ﬂies count to two? Trends Genet. 9, 385–390. 817. Cline, T.W. and Meyer, B.J. (1996). Vive la diff´erence: Males vs. females in ﬂies vs. worms. Annu. Rev. Genet. 30, 637– 702. 818. Clyne, P.J., Certel, S.J., de Bruyne, M., Zaslavsky, L., Johnson, W.A., and Carlson, J.R. (1999). The odor speciﬁcities of a subset of olfactory receptor neurons are governed by Acj6, a POU-domain transcription factor. Neuron 22, 339–347. 819. Clyne, P.J., Warr, C.G., Freeman, M.R., Lessing, D., Kim, J., and Carlson, J.R. (1999). A novel family of divergent seventransmembrane proteins: candidate odorant receptors in Drosophila. Neuron 22, 327–338. 820. Cobb, M. (1995). The ﬂy of the lords. Evolution 49, 581– 583. 821. Cobb, M. (1999). What and how do maggots smell? Biol. Rev. 74, 425–459. 822. Cobb, M.H. and Goldsmith, E.J. (1995). How MAP kinases are regulated. J. Biol. Chem. 270 #25, 14843–14846.

330

823. Cobb, M.H. and Goldsmith, E.J. (2000). Dimerization in MAP-kinase signaling. Trends Biochem. Sci. 25, 7–9. 824. Codd, E.F. (1968). “Cellular Automata.” Acad. Pr., New York. 825. Coen, E. (1999). “The Art of Genes: How Organisms Make Themselves.” Oxford Univ. Pr., New York. 826. Cohen, B., McGufﬁn, M.E., Pfeiﬂe, C., Segal, D., and Cohen, S.M. (1992). apterous, a gene required for imaginal disc development in Drosophila encodes a member of the LIM family of developmental regulatory proteins. Genes Dev. 6, 715–729. 827. Cohen, B., Simcox, A.A., and Cohen, S.M. (1993). Allocation of the thoracic imaginal primordia in the Drosophila embryo. Development 117, 597–608. 828. Cohen, B., Wimmer, E.A., and Cohen, S.M. (1991). Early development of leg and wing primordia in the Drosophila embryo. Mechs. Dev. 33, 229–240. 829. Cohen, G.B., Ren, R., and Baltimore, D. (1995). Modular binding domains in signal transduction proteins. Cell 80, 237–248. 830. Cohen, P.T.W., Brewis, N.D., Hughes, V., and Mann, D.J. (1990). Protein serine/threonine phosphatases: an expanding family. FEBS Letters 268, 355–359. 831. Cohen, S. and Hyman, A.A. (1994). When is a determinant a determinant? Curr. Biol. 5, 420–422. ¨ 832. Cohen, S. and Jurgens, G. (1991). Drosophila headlines. Trends Genet. 7, 267–272. 833. Cohen, S.M. (1990). Speciﬁcation of limb development in the Drosophila embryo by positional cues from segmentation genes. Nature 343, 173–177. 834. Cohen, S.M. (1993). Imaginal disc development. In “The Development of Drosophila melanogaster,” Vol. 2 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Lab. Pr.: Plainview, N. Y., pp. 747–841. 835. Cohen, S.M. (1996). Controlling growth of the wing: Vestigial integrates signals from the compartment boundaries. BioEssays 18, 855–858. ¨ ¨ 836. Cohen, S.M., Br¨onner, G., Kuttner, F., Jurgens, G., and J¨ackle, H. (1989). Distal-less encodes a homoeodomain protein required for limb development in Drosophila. Nature 338, 432–434. 837. Cohen, S.M. and Di Nardo, S. (1993). wingless: from embryo to adult. Trends Genet. 9, 189–192. ¨ 838. Cohen, S.M. and Jurgens, G. (1989). Proximal-distal pattern formation in Drosophila: cell autonomous requirement for Distal-less gene activity in limb development. EMBO J. 8, 2045–2055. ¨ 839. Cohen, S.M. and Jurgens, G. (1989). Proximal-distal pattern formation in Drosophila: graded requirement for Distal-less gene activity during limb development. Roux’s Arch. Dev. Biol. 198, 157–169. 840. Cole, E.S. and Palka, J. (1982). The pattern of campaniform sensilla on the wing and haltere of Drosophila melanogaster and several of its homeotic mutants. J. Embryol. Exp. Morph. 71, 41–61. 841. Coleman, J.E. (1992). Zinc proteins: Enzymes, storage proteins, transcription factors, and replication proteins. Annu. Rev. Biochem. 61, 897–946. 842. Coleman, K.G., Poole, S.J., Weir, M.P., Soeller, W.C., and Kornberg, T. (1987). The invected gene of Drosophila: sequence analysis and expression studies reveal a close kinship to the engrailed gene. Genes Dev. 1, 19– 28. 843. Colledge, M., Dean, R.A., Scott, G.K., Langeberg, L.K., Huganir, R.L., and Scott, J.D. (2000). Targeting of PKA to

REFERENCES

844. 845.

846.

847.

848.

849.

850.

851.

852.

853.

854.

855. 856.

857.

858. 859.

860.

861.

glutamate receptors through a MAGUK-AKAP complex. Neuron 27, 107–119. Colley, N.J. (2000). Actin’ up with Rac1. Science 290, 1902– 1903. Collier, C.P., Wong, E.W., Belohradsky, M., Raymo, F.M., Stoddart, J.F., Kuekes, P.J., Williams, R.S., and Heath, J.R. (1999). Electrically conﬁgurable molecular-based logic gates. Science 285, 391–394. Collier, S. and Gubb, D. (1997). Drosophila tissue polarity requires the cell-autonomous activity of the fuzzy gene, which encodes a novel transmembrane protein. Development 124, 4029–4037. Collins, M.K.L., Perkins, G.R., Rodriguez-Tarduchy, G., Nieto, M.A., and L´opez-Rivas, A. (1994). Growth factors as survival factors: regulation of apoptosis. BioEssays 16, 133–138. Collins, R.T., Furukawa, T., Tanese, N., and Treisman, J. (1999). Osa associates with the Brahma chromatin remodeling complex and promotes the activation of some target genes. EMBO J. 18, 7029–7040. Collins, R.T. and Treisman, J.E. (2000). Osa-containing Brahma chromatin remodeling complexes are required for the repression of Wingless target genes. Genes Dev. 14, 3140–3152. Combs, J.D. (1937). Genetic and environmental factors affecting the development of the sex-combs of Drosophila melanogaster. Genetics 22, 427–433. Condic, M.L., Fristrom, D., and Fristrom, J.W. (1991). Apical cell shape changes during Drosophila imaginal leg disc elongation: a novel morphogenetic mechanism. Development 111, 23–33. Condie, J.M. and Brower, D.L. (1989). Allelic interactions at the engrailed locus of Drosophila: engrailed protein expression in imaginal discs. Dev. Biol. 135, 31–42. Condron, B.G., Patel, N.H., and Zinn, K. (1994). engrailed controls glial/neuronal cell fate decisions at the midline of the central nervous system. Neuron 13, 541–554. Conley, C.A., Silburn, R., Singer, M.A., Ralston, A., RohwerNutter, D., Olson, D.J., Gelbart, W., and Blair, S.S. (2000). Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila. Development 127, 3947–3959. Conlon, I. and Raff, M. (1999). Size control in animal development. Cell 96, 235–244. Conlon, R.A., Reaume, A.G., and Rossant, J. (1995). Notch1 is required for the coordinate segmentation of somites. Development 121, 1533–1545. Connolly, J.P., Augustine, J.G., and Francklyn, C. (1999). Mutational analysis of the engrailed homeodomain recognition helix by phage display. Nucl. Acids Res. 27, 1182– 1189. Conrad, M. (1990). The geometry of evolution. BioSystems 24, 61–81. Cook, D., Fry, M.J., Hughes, K., Sumathipala, R., Woodgett, J.R., and Dale, T.C. (1996). Wingless inactivates glycogen synthase kinase-3 via an intracellular signalling pathway which involves a protein kinase C. EMBO J. 15, 4526–4536. Cook, T. and Desplan, C. (2001). Identiﬁcation of the homeodomain protein Prospero as a regulator of rhodopsin gene expression in the adult Drosophila eye. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a122. [See also 2001 Sems. Cell Dev. Biol. 12, 509–518.] Cook, T.A. (1914). “The Curves of Life.” Constable, London.

REFERENCES

862. Cooke, J. (1975). The emergence and regulation of spatial organization in early animal development. Annu. Rev. Biophys. Bioeng. 4, 185–217. 863. Cooke, J. (1978). Embryonic and regenerating patterns. Nature 271, 705–706. 864. Cooke, J. (1988). The early embryo and the formation of body pattern. Am. Sci. 76, 35–41. 865. Cooke, J. (1995). Morphogens in vertebrate development: how do they work? BioEssays 17, 93–96. [See also 2001 references: Curr. Biol. 11, R851–R854; Nature Rev. Genet. 2, 620–630.] 866. Cooke, J., Nowak, M.A., Boerlijst, M., and Maynard-Smith, J. (1997). Evolutionary origins and maintenance of redundant gene expression during metazoan development. Trends Genet. 13, 360–363. 867. Cooke, J. and Smith, J.C. (1990). Measurement of developmental time by cells of early embryos. Cell 60, 891–894. 868. Cooke, J. and Zeeman, E.C. (1976). A clock and wavefront model for control of the number of repeated structures during animal morphogenesis. J. Theor. Biol. 58, 455–476. [See also 2001 Nature 412, 780–781.] 869. Cooper, M.T.D. and Bray, S.J. (1999). Frizzled regulation of Notch signalling polarizes cell fate in the Drosophila eye. Nature 397, 526–530. 870. Cooper, M.T.D. and Bray, S.J. (2000). R7 photoreceptor speciﬁcation requires Notch activity. Curr. Biol. 10, 1507– 1510. 871. Cooper, M.T.D., Tyler, D.M., Furriols, M., Chalkiadaki, A., Delidakis, C., and Bray, S. (2000). Spatially restricted factors cooperate with Notch in the regulation of Enhancer of split genes. Dev. Biol. 221, 390–403. 872. Copeland, B.J. and Proudfoot, D. (1999). Alan Turing’s forgotten ideas in computer science. Sci. Am. 280 #4, 98–103. 873. Copeland, J.W.R., Nasiadka, A., Dietrich, B.H., and Krause, H.M. (1996). Patterning of the Drosophila embryo by a homeodomain-deleted Ftz polypeptide. Nature 379, 162– 165. [See also 2001 Genes Dev. 15, 1716–1723.] 874. Corfas, G. and Dudai, Y. (1990). Adaptation and fatigue of a mechanosensory neuron in wild-type Drosophila and in memory mutants. J. Neurosci. 10, 491–499. 875. Cornell, M., Evans, D.A.P., Mann, R., Fostier, M., Flasza, M., Monthatong, M., Artavanis-Tsakonas, S., and Baron, M. (1999). The Drosophila melanogaster Suppressor of deltex gene, a regulator of the Notch receptor signaling pathway, is an E3 Class ubiquitin ligase. Genetics 152, 567–576. 876. Cornell, R.A. and Kimelman, D. (1994). Combinatorial signaling in development. BioEssays 16, 577–581. 877. Coulter, D.E., Swaykus, E.A., Beran-Koehn, M.A., Goldberg, D., Wieschaus, E., and Schedl, P. (1990). Molecular analysis of odd-skipped, a zinc ﬁnger encoding segmentation gene with a novel pair-rule expression pattern. EMBO J. 8, 3795–3804. 878. Coulter, D.E. and Wieschaus, E. (1988). Gene activities and segmental patterning in Drosophila: analysis of odd-skipped and pair-rule double mutants. Genes Dev. 2, 1812–1823. 879. Courey, A.J. (2001). Cooperativity in transcriptional control. Curr. Biol. 10, R250–R252. 880. Couso, J.P., Bate, M., and Mart´ınez-Arias, A. (1993). A wingless-dependent polar coordinate system in Drosophila imaginal discs. Science 259, 484–489. 881. Couso, J.P. and Bishop, S.A. (1998). Proximo-distal development in the legs of Drosophila. Int. J. Dev. Biol. 42, 345– 352.

331

882. Couso, J.P., Bishop, S.A., and Martinez Arias, A. (1994). The wingless signalling pathway and the patterning of the wing margin in Drosophila. Development 120, 621–636. [See also 2001 Dev. Biol. 234, 13–23.] 883. Couso, J.P., Galindo, M.I., and Greig, S. (2001). Intercalation of proximal-distal fates independently of wg and dpp. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a196. 884. Couso, J.P. and Gonz´alez-Gait´an, M. (1993). Embryonic limb development in Drosophila. Trends Genet. 9, 371– 373. 885. Couso, J.P., Knust, E., and Martinez Arias, A. (1995). Serrate and wingless cooperate to induce vestigial gene expression and wing formation in Drosophila. Curr. Biol. 5, 1437–1448. 886. Couso, J.P. and Martinez Arias, A. (1994). Notch is required for wingless signaling in the epidermis of Drosophila. Cell 79, 259–272. 887. Cowan, W.M. (1973). Neuronal death as a regulative mechanism in the control of cell number in the nervous system. In “Development and Aging in the Nervous System,” (M. Rockstein and M.L. Sussman, eds.). Acad. Pr.: New York, pp. 19–41. 888. Cowan, W.M., Fawcett, J.W., O’Leary, D.D.M., and Stanﬁeld, B.B. (1984). Regressive events in neurogenesis. Science 225, 1258–1265. 889. Cowman, A.F., Zuker, C.S., and Rubin, G.M. (1986). An opsin gene expressed in only one photoreceptor cell type of the Drosophila eye. Cell 44, 705–710. 890. Cox, R.T., Kirkpatrick, C., and Peifer, M. (1996). Armadillo is required for adherens junction assembly, cell polarity, and morphogenesis during Drosophila embryogenesis. J. Cell Biol. 134, 133–148. 891. Cox, R.T., McEwen, D.G., Myster, D.L., Duronio, R.J., Loureiro, J., and Peifer, M. (2000). A screen for mutations that suppress the phenotype of Drosophila armadillo, the β-catenin homolog. Genetics 155, 1725–1740. 892. Cox, R.T., Pai, L.-M., Kirkpatrick, C., Stein, J., and Peifer, M. (1999). Roles of the C terminus of Armadillo in Wingless signaling in Drosophila. Genetics 153, 319–332. 893. Cox, R.T., Pai, L.-M., Miller, J.R., Orsulic, S., Stein, J., McCormick, C.A., Audeh, Y., Wang, W., Moon, R.T., and Peifer, M. (1999). Membrane-tethered Drosophila Armadillo cannot transduce Wingless signal on its own. Development 126, 1327–1335. 894. Cox, R.T. and Peifer, M. (1998). Wingless signaling: The inconvenient complexities of life. Curr. Biol. 8, R140– R144. 895. Coyle-Thompson, C.A. and Banerjee, U. (1993). The strawberry notch gene functions with Notch in common developmental pathways. Development 119, 377–395. [See also 2001 Mechs. Dev. 109, 241–251.] 896. Craven, S.E. and Bredt, D.S. (1998). PDZ proteins organize synaptic signaling pathways. Cell 93, 495–498. 897. Crew, J.R., Batterham, P., and Pollock, J.A. (1997). Developing compound eye in lozenge mutants of Drosophila: lozenge expression in the R7 equivalence group. Dev. Genes Evol. 206, 481–493. 898. Crews, C.M. and Erikson, R.L. (1993). Extracellular signals and reversible protein phosphorylation: what to Mek of it all. Cell 74, 215–217. 899. Crews, S.T. (1998). Control of cell lineage-speciﬁc development and transcription by bHLH-PAS proteins. Genes Dev. 12, 607–620. 900. Crews, S.T. and Fan, C.-M. (1999). Remembrance of things

332

901.

902. 903. 904.

905.

906.

907.

908.

909.

910.

911.

912.

913.

914.

915.

916.

917.

REFERENCES

PAS: regulation of development by bHLH-PAS proteins. Curr. Opin. Gen. Dev. 9, 580–587. Crews, S.T., Thomas, J.B., and Goodman, C.S. (1988). The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product. Cell 52, 143–151. Crick, F. (1970). Diffusion in embryogenesis. Nature 225, 420–422. Crick, F.H.C. (1971). The scale of pattern formation. Symp. Soc. Exp. Biol. 25, 429–438. Crick, F.H.C. and Lawrence, P.A. (1975). Compartments and polyclones in insect development. Science 189, 340– 347. Cronmiller, C. and Cummings, C.A. (1993). The daughterless gene product in Drosophila is a nuclear protein that is broadly expressed throughout the organism during development. Mechs. Dev. 42, 159–169. [See also 2001 Development 128, 4705–4714.] Cronmiller, C., Schedl, P., and Cline, T.W. (1988). Molecular characterization of daughterless, a Drosophila sex determination gene with multiple roles in development. Genes Dev. 2, 1666–1676. Crosby, M.A., Lundquist, E.A., Tautvydas, R.M., and Johnson, J.J. (1993). The 3 regulatory region of the AbdominalB gene: genetic analysis supports a model of reiterated and interchangeable regulatory elements. Genetics 134, 809–824. Crosby, M.A., Miller, C., Alon, T., Watson, K.L., Verrijzer, C.P., Goldman-Levi, R., and Zak, N.B. (1999). The trithorax group gene moira encodes a Brahma-associated putative chromatin-remodeling factor in Drosophila melanogaster. Mol. Cell. Biol. 19, 1159–1170. Crozatier, M., Valle, D., Dubois, L., Ibnsouda, S., and Vincent, A. (1996). collier, a novel regulator of Drosophila head development, is expressed in a single mitotic domain. Curr. Biol. 6, 707–718. Crozatier, M. and Vincent, A. (1999). Requirement for the Drosophila COE transcription factor Collier in formation of an embryonic muscle: transcriptional response to Notch signalling. Development 126, 1495–1504. [See also 2001 Mechs. Dev. 108, 3–12.] Cubadda, Y., Heitzler, P., Ray, R.P., Bourouis, M., Ramain, P., Gelbart, W., Simpson, P., and Haenlin, M. (1997). u-shaped encodes a zinc-ﬁnger protein that regulates the proneural genes achaete and scute during the formation of bristles in Drosophila. Genes Dev. 11, 3083–3095. Cubas, P., de Celis, J.-F., Campuzano, S., and Modolell, J. (1991). Proneural clusters of achaete-scute expression and the generation of sensory organs in the Drosophila imaginal wing disc. Genes Dev. 5, 996–1008. Cubas, P. and Modolell, J. (1992). The extramacrochaetae gene provides information for sensory organ patterning. EMBO J. 11, 3385–3393. Cubas, P., Modolell, J., and Ruiz-G´omez, M. (1994). The helix-loop-helix extramacrochaetae protein is required for proper speciﬁcation of many cell types in the Drosophila embryo. Development 120, 2555–2565. Cuenoud, B. and Schepartz, A. (1993). Altered speciﬁcity of DNA-binding proteins with transition metal dimerization domains. Science 259, 510–513. Cui, X. and Doe, C.Q. (1995). The role of the cell cycle and cytokinesis in regulating neuroblast sublineage gene expression in the Drosophila CNS. Development 121, 3233– 3243. Cul´ı, J., Mart´ın-Blanco, E., and Modolell, J. (2001). The EGF

918.

919.

920.

921.

922.

923.

924.

925.

926.

927.

928.

929. 930.

931.

932.

933.

934.

935.

receptor and N signalling pathways act antagonistically in Drosophila mesothorax bristle patterning. Development 128, 299–308. Cul´ı, J. and Modolell, J. (1998). Proneural gene selfstimulation in neural precursors: an essential mechanism for sense organ development that is regulated by Notch signaling. Genes Dev. 12, 2036–2047. Cullen, C.F. and Milner, M.J. (1991). Parameters of growth in primary cultures and cell lines established from Drosophila imaginal discs. Tissue Cell 23, 29–39. Cumberledge, S. and Krasnow, M.A. (1993). Intercellular signalling in Drosophila segment formation reconstructed in vitro. Nature 363, 549–552. Cumberledge, S. and Reichsman, F. (1997). Glycosaminoglycans and WNTs: just a spoonful of sugar helps the signal do down. Trends Genet. 13, 421–423. Cummings, F.W. and Prothero, J.W. (1978). A model of pattern formation in multicellular organisms. Collective Phenom. 3, 41–53. Cummins, H. and Midlo, C. (1943). “Finger Prints, Palms and Soles. An Introduction to Dermatoglyphics.” Dover, New York. Cunningham, G.N., Smith, N.M., Makowski, M.K., and Kuhn, D.T. (1983). Enzyme patterns in D. melanogaster imaginal discs: Distribution of glucose-6-phosphate and 6-phospho-gluconate dehydrogenase. Mol. Gen. Genet. 191, 238–243. Curcio, C.A., Sloan, K.R., Jr., Packer, O., Hendrickson, A.E., and Kalina, R.E. (1987). Distribution of cones in human and monkey retina: individual variability and radial asymmetry. Science 236, 579–582. Curtin, K., D., Huang, Z.J., and Rosbash, M. (1995). Temporally regulated nuclear entry of the Drosophila period protein contributes to the circadian clock. Neuron 14, 365–372. Curtiss, J. and Heilig, J.S. (1995). Establishment of Drosophila imaginal precursor cells is controlled by the Arrowhead gene. Development 121, 3819–3825. Curtiss, J. and Heilig, J.S. (1997). Arrowhead encodes a LIM homeodomain protein that distinguishes subsets of Drosophila imaginal cells. Dev. Biol. 190, 129–141. Curtiss, J. and Heilig, J.S. (1998). DeLIMiting development. BioEssays 20, 58–69. Curtiss, J. and Mlodzik, M. (2000). Morphogenetic furrow initiation and progression during eye development in Drosophila: the roles of decapentaplegic, hedgehog, and eyes absent. Development 127, 1325–1336. Cutforth, T. and Gaul, U. (1997). The genetics of visual system development in Drosophila: speciﬁcation, connectivity and asymmetry. Curr. Opin. Neurobiol. 7, 48– 54. Cutforth, T. and Rubin, G.M. (1994). Mutations in Hsp83 and cdc37 impair signaling by the Sevenless receptor tyrosine kinase in Drosophila. Cell 77, 1027–1036. Czerny, T., Halder, G., Kloter, U., Souabni, A., Gehring, W.J., and Busslinger, M. (1999). twin of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development. Molec. Cell 3, 297–307. Czerny, T., Schaffner, G., and Busslinger, M. (1993). DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site. Genes Dev. 7, 2048–2061. D’Avino, P.P. and Thummel, C.S. (1998). crooked legs encodes a family of zinc ﬁnger proteins required for leg morphogenesis and ecdysone-regulated gene expression

REFERENCES

936.

937.

938.

939.

940. 941.

942.

943.

944.

945. 946.

947.

948.

949.

950.

951.

952. 953.

during Drosophila metamorphosis. Development 125, 1733–1745. D’Avino, P.P. and Thummel, C.S. (2000). The ecdysone regulatory pathway controls wing morphogenesis and integrin expression during Drosophila metamorphosis. Dev. Biol. 220, 211–224. D’Costa, A.R., Reifegerste, R., Siera, S., and Moses, K. (2001). Characterisation of a dominant modiﬁer of hedgehog. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a271. Daga, A., Karlovich, C.A., Dumstrei, K., and Banerjee, U. (1996). Patterning of cells in the Drosophila eye by Lozenge, which shares homologous domains with AML1. Genes Dev. 10, 1194–1205. Dahanukar, A. and Wharton, R.P. (1996). The Nanos gradient in Drosophila embryos is generated by translational regulation. Genes Dev. 10, 2610–2620. Dahl, E., Koseki, H., and Balling, R. (1997). Pax genes and organogenesis. BioEssays 19, 755–765. Dahlsveen, I.K. and McNeill, H. (2001). Identiﬁcation and characterisation of proteins that interact wtih the transcription factor Mirror. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a123. Dahmann, C. and Basler, K. (1999). Compartment boundaries at the edge of development. Trends Genet. 15, 320– 326. Dahmann, C. and Basler, K. (2000). Opposing transcriptional outputs of Hedgehog signaling and Engrailed control compartmental cell sorting at the Drosophila A/P boundary. Cell 100, 411–422. Dai, H., Hogan, C., Gopalakrishnan, B., Torres-Vazquez, J., Nguyen, M., Park, S., Raftery, L.A., Warrior, R., and Arora, K. (2000). The zinc ﬁnger protein Schnurri acts as a Smad partner in mediating the transcriptional response to Decapentaplegic. Dev. Biol. 227, 373–387. Dale, K.J. and Pourqui´e, O. (2000). A clock-work somite. BioEssays 22, 72–83. [See also 2001 J. Anat. 199, 169–175.] Dale, L. and Bownes, M. (1980). Is regeneration in Drosophila the result of epimorphic regulation? W. Roux’s Arch. 189, 91–96. Dale, L. and Bownes, M. (1985). Pattern regulation in fragments of Drosophila wing discs which show variable wound healing. J. Embryol. Exp. Morph. 85, 95–109. Dambly-Chaudi`ere, C. and Ghysen, A. (1987). Independent subpatterns of sense organs require independent genes of the achaete-scute complex in Drosophila larvae. Genes Dev. 1, 297–306. Dambly-Chaudi`ere, C., Ghysen, A., Jan, L.Y., and Jan, Y.N. (1988). The determination of sense organs in Drosophila: interaction of scute with daughterless. Roux’s Arch. Dev. Biol. 197, 419–423. Dambly-Chaudi`ere, C., Jamet, E., Burri, M., Bopp, D., Basler, K., Hafen, E., Dumont, N., Spielmann, P., Ghysen, A., and Noll, M. (1992). The paired box gene pox neuro: a determinant of poly-innervated sense organs in Drosophila. Cell 69, 159–172. Dambly-Chaudi`ere, C. and Leyns, L. (1992). The determination of sense organs in Drosophila: a search for interacting genes. Int. J. Dev. Biol. 36, 85–91. Dambly-Chaudi`ere, C. and Vervoort, M. (1998). The bHLH genes in neural development. Int. J. Dev. Biol. 42, 269–273. Damelin, M. and Silver, P.A. (2000). Mapping interactions between nuclear transport factors in living cells reveals pathways through the nuclear pore complex. Molec. Cell 5, 133–140.

333

954. Damke, H. (1996). Dynamin and receptor-mediated endocytosis. FEBS Letters 389, 48–51. 955. Daniel, A., Dumstrei, K., Lengyel, J.A., and Hartenstein, V. (1999). The control of cell fate in the embryonic visual system by atonal, tailless and EGFR signaling. Development 126, 2945–2954. 956. Daniel, J.M. and Reynolds, A.B. (1997). Tyrosine phosphorylation and cadherin/catenin function. BioEssays 19, 883–891. 957. Danpure, C.J. (1995). How can the products of a single gene be localized to more than one intracellular compartment? Trends Cell Biol. 5, 230–238. 958. Darnell, J.E., Jr. (1997). STATs and gene regulation. Science 277, 1630–1635. 959. Das, P., Maduzia, L.L., Wang, H., Finelli, A.L., Cho, S.-H., Smith, M.M., and Padgett, R.W. (1998). The Drosophila gene Medea demonstrates the requirement for different classes of Smads in dpp signaling. Development 125, 1519– 1528. 960. Dasen, J.S. and Rosenfeld, M.G. (1999). Combinatorial codes in signaling and synergy: lessons from pituitary development. Curr. Opin. Gen. Dev. 9, 566–574. 961. Datar, S.A., Jacobs, H.W., de la Cruz, A.F.A., Lehner, C.F., and Edgar, B.A. (2000). The Drosophila Cyclin D-Cdk4 complex promotes cellular growth. EMBO J. 19, 4543– 4554. 962. Datta, R.K. and Mukherjee, A.S. (1971). Developmental genetics of the mutant combgap in Drosophila melanogaster. I. Effect on the morphology and chaetotaxy of the prothoracic leg. Genetics 68, 269–286. 963. Datta, S., Stark, K., and Kankel, D.R. (1993). Enhancer detector analysis of the extent of genomic involvement in nervous system development in Drosophila melanogaster. J. Neurobiol. 24, 824–841. 964. Daubresse, G., Deuring, R., Moore, L., Papoulas, O., Zakrajsek, I., Waldrip, W.R., Scott, M.P., Kennison, J.A., and Tamkun, J.W. (1999). The Drosophila kismet gene is related to chromatin-remodeling factors and is required for both segmentation and segment identity. Development 126, 1175–1187. 965. Daum, G., Eisenmann-Tappe, I., Fries, H.-W., Troppmair, J., and Rapp, U.R. (1994). The ins and outs of Raf kinases. Trends Biochem. Sci. 19, 474–480. 966. David, G. (1993). Integral membrane heparan sulfate proteoglycans. FASEB J. 7, 1023–1030. 967. Davidson, E.H. (1993). Later embryogenesis: regulatory circuitry in morphogenetic ﬁelds. Development 118, 665– 690. 968. Davidson, E.H. (2001). “Genomic Regulatory Systems: Development and Evolution.” Acad. Pr., New York. 969. Davies, E., Hsiao, F., Williams, A., and Rebay, I. (2001). Eyes absent mediates cross-talk between a network of retinal determination genes and the RTK/Ras/MAPK signaling pathway. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a55. [See also 2001 Dev. Cell 1, 51–61.] 970. Davis, C.G. (1990). The many faces of epidermal growth factor repeats. New Biol. 2, 410–419. 971. Davis, R.J. (2000). Signal transduction by the JNK group of MAP kinases. Cell 103, 239–252. 972. Dawid, I.B. (1998). LIM protein interactions: Drosophila enters the stage. Trends Genet. 14, 480–482. 973. Dawid, I.B., Breen, J.J., and Toyama, R. (1998). LIM domains: multiple roles as adapters and functional modiﬁers in protein interactions. Trends Genet. 14, 156– 162.

334

974. Dawkins, R. (1996). “Climbing Mount Improbable.” Norton, N.Y. 975. Dawson, I.A., Roth, S., and Artavanis-Tsakonas, S. (1995). The Drosophila cell cycle gene ﬁzzy is required for normal degradation of cyclins A and B during mitosis and has homology to the CDC20 gene of Saccharomyces cerevisiae. J. Cell Biol. 129, 725–737. 976. Dawson, S.R., Turner, D.L., Weintraub, H., and Parkhurst, S.M. (1995). Speciﬁcity for the Hairy/Enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression. Mol. Cell. Biol. 15, 6923– 6931. 977. Day, S.J. and Lawrence, P.A. (2000). Measuring dimensions: the regulation of size and shape. Development 127, 2977–2987. 978. de Beer, G. (1958). “Embryos and Ancestors.” 3rd ed. Clarendon Pr., Oxford. 979. de Beer, T., Carter, R.E., Lobel-Rice, K.E., Sorkin, A., and Overduin, M. (1998). Structure and Asn-Pro-Phe binding pocket of the Eps15 homology domain. Science 281, 1357– 1360. 980. de Celis, J.F. (1997). Expression and function of decapentaplegic and thick veins during the differentiation of the veins in the Drosophila wing. Development 124, 1007– 1018. 981. de Celis, J.F. (1998). Positioning and differentiation of veins in the Drosophila wing. Int. J. Dev. Biol. 42, 335–343. 982. de Celis, J.F. (1999). The function of vestigial in Drosophila wing development: how are tissue-speciﬁc responses to signalling pathways speciﬁed? BioEssays 21, 542–545. 983. de Celis, J.F., Baonza, A., and Garc´ıa-Bellido, A. (1995). Behavior of extramacrochaetae mutant cells in the morphogenesis of the Drosophila wing. Mechs. Dev. 53, 209–221. 984. de Celis, J.F. and Barrio, R. (2000). Function of the spalt/ spalt-related gene complex in positioning the veins in the Drosophila wing. Mechs. Dev. 91, 31–41. 985. de Celis, J.F., Barrio, R., del Arco, A., and Garc´ıa-Bellido, A. (1993). Genetic and molecular characterization of a Notch mutation in its Delta- and Serrate-binding domain in Drosophila. Proc. Natl. Acad. Sci. USA 90, 4037–4041. 986. de Celis, J.F., Barrio, R., and Kafatos, F.C. (1996). A gene complex acting downstream of dpp in Drosophila wing morphogenesis. Nature 381, 421–424. 987. de Celis, J.F., Barrio, R., and Kafatos, F.C. (1999). Regulation of the spalt/spalt-related gene complex and its function during sensory organ development in the Drosophila thorax. Development 126, 2653–2662. 988. de Celis, J.F. and Bray, S. (1997). Feed-back mechanisms affecting Notch activation at the dorsoventral boundary in the Drosophila wing. Development 124, 3241–3251. 989. de Celis, J.F., Bray, S., and Garcia-Bellido, A. (1997). Notch signalling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing. Development 124, 1919–1928. 990. de Celis, J.F. and Bray, S.J. (2000). The Abruptex domain of Notch regulates negative interactions between Notch, its ligands and Fringe. Development 127, 1291–1302. 991. de Celis, J.F., de Celis, J., Ligoxygakis, P., Preiss, A., Delidakis, C., and Bray, S. (1996). Functional relationships between Notch, Su(H) and the bHLH genes of the E(spl) complex: the E(spl) genes mediate only a subset of Notch activities during imaginal development. Development 122, 2719–2728. 992. de Celis, J.F. and Garcia-Bellido, A. (1994). Modiﬁcations of

REFERENCES

993.

994.

995.

996.

997.

998.

999.

1000.

1001.

1002.

1003.

1004.

1005.

1006.

1007.

1008.

1009.

the Notch function by Abruptex mutations in Drosophila melanogaster. Genetics 136, 183–194. de Celis, J.F. and Garc´ıa-Bellido, A. (1994). Roles of the Notch gene in Drosophila wing morphogenesis. Mechs. Dev. 46, 109–122. de Celis, J.F., Garcia-Bellido, A., and Bray, S.J. (1996). Activation and function of Notch at the dorsal-ventral boundary of the wing imaginal disc. Development 122, 359–369. de Celis, J.F., Llimargas, M., and Casanova, J. (1995). ventral veinless, the gene encoding the Cf1a transcription factor, links positional information and cell differentiation during embryonic and imaginal development in Drosophila melanogaster. Development 121, 3405– 3416. de Celis, J.F., Mar´ı-Beffa, M., and Garc´ıa-Bellido, A. (1991). Cell-autonomous role of Notch, an epidermal growth factor homologue, in sensory organ differentiation in Drosophila. Proc. Natl. Acad. Sci. USA 88, 632–636. de Celis, J.F., Mar´ı-Beffa, M., and Garc´ıa-Bellido, A. (1991). Function of trans-acting genes of the achaete-scute complex in sensory organ patterning in the mesonotum of Drosophila. Roux’s Arch. Dev. Biol. 200, 64–76. de Celis, J.F. and Ruiz-G´omez, M. (1995). groucho and hedgehog regulate engrailed expression in the anterior compartment of the Drosophila wing. Development 121, 3467–3476. de Celis, J.F., Tyler, D.M., de Celis, J., and Bray, S.J. (1998). Notch signalling mediates segmentation of the Drosophila leg. Development 125, 4617–4626. [See also 2001 Mechs. Dev. 105, 115–127.] de la Concha, A., Dietrich, U., Weigel, D., and CamposOrtega, J.A. (1988). Functional interactions of neurogenic genes of Drosophila melanogaster. Genetics 118, 499–508. De Lorenzo, C.M., Huwe, A.W., Spillane, M., and Bryant, P.J. (2001). The Dlg multimeric complex and its function in cell proliferation control. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a156. de Nooij, J.C., Graber, K.H., and Hariharan, I.K. (2000). Expression of the cyclin-dependent kinase inhibitor Dacapo is regulated by Cyclin E. Mechs. Dev. 97, 73–83. de Nooij, J.C. and Hariharan, I.K. (1995). Uncoupling cell fate determination from patterned cell division in the Drosophila eye. Science 270, 983–985. de Nooij, J.C., Letendre, M.A., and Hariharan, I.K. (1996). A cyclin-dependent kinase inhibitor, Dacapo, is necessary for timely exit from the cell cycle during Drosophila embryogenesis. Cell 87, 1237–1247. De Robertis, E.M., Oliver, G., and Wright, C.V.E. (1989). Determination of axial polarity in the vertebrate embryo: homeodomain proteins and homeogenetic induction. Cell 57, 189–191. de Vetten, N., Quattrocchio, F., Mol, J., and Koes, R. (1997). The an11 locus controlling ﬂower pigmentation in petunia encodes a novel WD-repeat protein conserved in yeast, plants, and animals. Genes Dev. 11, 1422–1434. de Zulueta, P., Alexandre, E., Jacq, B., and Kerridge, S. (1994). Homeotic complex and teashirt genes co-operate to establish trunk segmental identities in Drosophila. Development 120, 2278–2296. Deak, I.I. (1980). A model linking segmentation, compartmentalization and regeneration in Drosophila development. J. Theor. Biol. 84, 477–504. Dearden, P. and Akam, M. (2000). A role for Fringe in segment morphogenesis but not segment formation in the

REFERENCES

1010. 1011.

1012.

1013.

1014.

1015.

1016.

1017.

1018.

1019. 1020.

1021.

1022.

1023.

1024. 1025.

1026.

1027.

1028.

grasshopper, Shistocerca gregaria. Dev. Genes Evol. 210, 329–336. Dearden, P. and Akam, M. (2000). Segmentation in silico. Nature 406, 131–132. Dearolf, C.R., Topol, J., and Parker, C.S. (1989). The caudal gene product is a direct activator of fushi tarazu transcription during Drosophila embryogenesis. Nature 341, 340–343. DeCamillis, M., Cheng, N., Pierre, D., and Brock, H.W. (1992). The polyhomeotic gene of Drosophila encodes a chromatin protein that shares polytene chromosomebinding sites with Polycomb. Genes Dev. 6, 223–232. Decoville, M., Giacomello, E., Leng, M., and Locker, D. (2001). DSP1, an HMG-like protein, is involved in the regulation of homeotic genes. Genetics 157, 237–244. Dekker, N., Cox, M., Boelens, R., Verrijzer, C.P., van der Vliet, P.C., and Kaptein, R. (1993). Solution structure of the POU-speciﬁc DNA-binding domain of Oct-1. Nature 362, 852–855. Delattre, M., Briand, S., Paces-Fessy, M., and BlanchetTournier, M.-F. (1999). The Suppressor of fused gene, involved in Hedgehog signal transduction in Drosophila, is conserved in mammals. Dev. Genes Evol. 209, 294– 300. Delgado, R., Maureira, C., Oliva, C., Kidokoro, Y., and Labarca, P. (2000). Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire. Neuron 28, 941–953. Delidakis, C. and Artavanis-Tsakonas, S. (1992). The Enhancer of split [E(spl)] locus of Drosophila encodes seven independent helix-loop-helix proteins. Proc. Natl. Acad. Sci. USA 89, 8731–8735. Delidakis, C., Preiss, A., Hartley, D.A., and ArtavanisTsakonas, S. (1991). Two genetically and molecularly distinct functions involved in early neurogenesis reside within the Enhancer of split locus of Drosophila melanogaster. Genetics 129, 803–823. Demerec, M., ed. (1950). “Biology of Drosophila.” Hafner, New York. Demidenko, Z., Badenhorst, P., Jones, T., Bi, X., and Mortin, M.A. (2001). Regulated nuclear export of the homeodomain transcription factor Prospero. Development 128, 1359–1367. DeMille, M.M.C., Kimmel, B.E., and Rubin, G.M. (1996). A Drosophila gene regulated by rough and glass shows similarity to ena and VASP. Gene 183, 103–108. ¨ Denef, N., Neubuser, D., Perez, L., and Cohen, S.M. (2000). Hedgehog induces opposite changes in turnover and subcellular localization of Patched and Smoothened. Development 102, 521–531. Denell, R. (1994). Discovery and genetic deﬁnition of the Drosophila Antennapedia Complex. Genetics 138, 549– 552. Denell, R.E. (1978). Homoeosis in Drosophila: II. A genetic analysis of Polycomb. Genetics 90, 277–289. Denell, R.E., Brown, S.J., and Beeman, R.W. (1996). Evolution of the organization and function of insect homeotic complexes. Sems. Cell Dev. Biol. 7, 527–538. Denell, R.E. and Frederick, R.D. (1983). Homoeosis in Drosophila: a description of the Polycomb lethal syndrome. Dev. Biol. 97, 34–47. Derynck, R., Zhang, Y., and Feng, X.-H. (1998). Smads: transcriptional activators of TGF-β responses. Cell 95, 737–740. Deshpande, G., Stukey, J., and Schedl, P. (1995). scute (sis-

335

1029.

1030. 1031.

1032.

1033. 1034.

1035.

1036.

1037.

1038.

1039.

1040.

1041.

1042.

1043.

1044.

1045.

b) function in Drosophila sex determination. Mol. Cell. Biol. 15, 4430–4440. Deshpande, N., Chopra, A., Rangarajan, A., Shashidhara, L.S., Rodrigues, V., and Krishna, S. (1997). The human Transcription Enhancer Factor-1, TEF-1, can substitute for Drosophila scalloped during wingblade development. J. Biol. Chem. 272 #16, 10664–10668. Desplan, C. (1997). Eye development: governed by a dictator or a junta? Cell 91, 861–864. Desplan, C., Theis, J., and O’Farrell, P.H. (1985). The Drosophila developmental gene, engrailed, encodes a sequence-speciﬁc DNA binding activity. Nature 318, 630– 635. Devoe, R.D. (1985). The eye: electrical activity. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology,” Vol. 6 (G.A. Kerkut and L.I. Gilbert, eds.). Pergamon Pr.: Oxford, pp. 277–354. Dewdney, A.K. (1988). The hodgepodge machine makes waves. Sci. Am. 259 #2, 104–107. Dewdney, A.K. (1989). “The Turing Omnibus: 61 Excursions in Computer Science.” Computer Sci. Pr., Rockville, MD. Dhalluin, C., Yan, K.S., Plotnikova, O., Lee, K.W., Zeng, L., Kuti, M., Mujtaba, S., Goldfarb, M.P., and Zhou, M.-M. (2000). Structural basis of SNT PTB domain interactions with distinct neurotrophic receptors. Molec. Cell 6, 921– 929. Dhordain, P., Albagli, O., Lin, R.J., Ansieau, S., Quief, S., Leutz, A., Kerckaert, J.-P., Evans, R.M., and Leprince, D. (1997). Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein. Proc. Natl. Acad. Sci. USA 94, 10762–10767. Diaz-Benjumea, F.J., Cohen, B., and Cohen, S.M. (1994). Cell interaction between compartments establishes the proximal-distal axis of Drosophila legs. Nature 372, 175– 179. Diaz-Benjumea, F.J. and Cohen, S.M. (1993). Interaction between dorsal and ventral cells in the imaginal disc directs wing development in Drosophila. Cell 75, 741–752. Diaz-Benjumea, F.J. and Cohen, S.M. (1994). wingless acts through the shaggy/zeste-white 3 kinase to direct dorsalventral axis formation in the Drosophila leg. Development 120, 1661–1670. Diaz-Benjumea, F.J. and Cohen, S.M. (1995). Serrate signals through Notch to establish a Wingless-dependent organizer at the dorsal/ventral compartment boundary of the Drosophila wing. Development 121, 4215–4225. Diaz-Benjumea, F.J. and Garc´ıa-Bellido, A. (1990). Genetic analysis of the wing vein pattern of Drosophila. Roux’s Arch. Dev. Biol. 198, 336–354. D´ıaz-Benjumea, F.J. and Garc´ıa-Bellido, A. (1990). Behaviour of cells mutant for an EGF receptor homologue of Drosophila in genetic mosaics. Proc. Roy. Soc. Lond. B 242, 36–44. Diaz-Benjumea, F.J. and Hafen, E. (1994). The sevenless signalling cassette mediates Drosophila EGF receptor function during epidermal development. Development 120, 569–578. DiBello, P.R., Withers, D.A., Bayer, C.A., Fristrom, J.W., and Guild, G.M. (1991). The Drosophila Broad-Complex encodes a family of related proteins containing zinc ﬁngers. Genetics 129, 385–397. Dickinson, M.H., Lehmann, F.-O., and Sane, S.P. (1999). Wing rotation and the aerodynamic basis of insect ﬂight. Science 284, 1954–1960.

336

1046. Dickinson, W.J. (1988). On the architecture of regulatory systems: evolutionary insights and implications. BioEssays 8, 204–208. 1047. Dickson, B. (1995). Nuclear factors in sevenless signalling. Trends Genet. 11, 106–111. 1048. Dickson, B. and Hafen, E. (1993). Genetic dissection of eye development in Drosophila. In “The Development of Drosophila melanogaster,” Vol. 2 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Lab. Pr.: Plainview, N. Y., pp. 1327–1362. 1049. Dickson, B., Sprenger, F., and Hafen, E. (1992). Prepattern in the developing Drosophila eye revealed by an activated torso-sevenless chimeric receptor. Genes Dev. 6, 2327–2339. 1050. Dickson, B., Sprenger, F., Morrison, D., and Hafen, E. (1992). Raf functions downstream of Ras1 in the Sevenless signal transduction pathway. Nature 360, 600–603. 1051. Dickson, B.J. (1998). Photoreceptor development: breaking down the barriers. Curr. Biol. 8, R90–R92. 1052. Dickson, B.J., Dom´ınguez, M., van der Straten, A., and Hafen, E. (1995). Control of Drosophila photoreceptor cell fates by Phyllopod, a novel nuclear protein acting downstream of the Raf kinase. Cell 80, 453–462. 1053. Diederich, R.J., Matsuno, K., Hing, H., and ArtavanisTsakonas, S. (1994). Cytosolic interaction between deltex and Notch ankyrin repeats implicates deltex in the Notch signaling pathway. Development 120, 473–481. 1054. Dierick, H. and Bejsovec, A. (1999). Cellular mechanisms of Wingless/Wnt signal transduction. Curr. Topics Dev. Biol. 43, 153–190. 1055. Dierick, H.A. and Bejsovec, A. (1998). Functional analysis of Wingless reveals a link between intercellular ligand transport and dorsal-cell-speciﬁc signaling. Development 125, 4729–4738. 1056. Dietrich, M.R. (2000). From hopeful monsters to homeotic effects: Richard Goldschmidt’s integration of development, evolution, and genetics. Amer. Zool. 40, 738–747. 1057. Dietrich, U. and Campos-Ortega, J.A. (1980). The effect of temperature on shibire ts cell clones in the compound eye of Drosophila melanogaster. W. Roux’s Arch. 188, 55–63. 1058. Dietrich, U. and Campos-Ortega, J.A. (1984). The expression of neurogenic loci in imaginal epidermal cells of Drosophila melanogaster. J. Neurogen. 1, 315–332. 1059. Dietrich, W. (1909). Die Facettenaugen der Dipteren. Z. Wiss. Zool. 92, 465–539. 1060. Diez del Corral, R., Aroca, P., G´omez-Skarmeta, J.L., Cavodeassi, F., and Modolell, J. (1999). The Iroquois homeodomain proteins are required to specify body wall identity in Drosophila. Genes Dev. 13, 1754–1761. 1061. Dimitratos, S.D., Woods, D.F., and Bryant, P.J. (1997). Camguk, Lin-2, and CASK: novel membrane-associated guanylate kinase homologs that also contain CaM kinase domains. Mechs. Dev. 63, 127–130. 1062. Dimitratos, S.D., Woods, D.F., Stathakis, D.G., and Bryant, P.J. (1999). Signaling pathways are focused at specialized regions of the plasma membrane by scaffolding proteins of the MAGUK family. BioEssays 21, 912–921. [See also 2001 Trends Genet. 17, 511–519.] 1063. DiNardo, S., Heemskerk, J., Dougan, S., and O’Farrell, P.H. (1994). The making of a maggot: patterning the Drosophila embryonic epidermis. Curr. Opinion Gen. Dev. 4, 529–534. 1064. DiNardo, S., Kuner, J.M., Theis, J., and O’Farrell, P.H. (1985). Development of embryonic pattern in D. melanogaster as revealed by accumulation of the nuclear engrailed protein. Cell 43, 59–69.

REFERENCES

1065. DiNardo, S., Sher, E., Heemskerk-Jongens, J., Kassis, J.A., and O’Farrell, P.H. (1988). Two-tiered regulation of spatially patterned engrailed gene expression during Drosophila embryogenesis. Nature 332, 604–609. 1066. Dingwall, C. and Laskey, R.A. (1998). Nuclear import: a tale of two sites. Curr. Biol. 8, R922–R924. 1067. Djian, P. (1998). Evolution of simple repeats in DNA and their relation to human disease. Cell 94, 155–160. 1068. Doctor, J.S., Jackson, P.D., Rashka, K.E., Visalli, M., and Hoffmann, F.M. (1992). Sequence, biochemical characterization, and developmental expression of a new member of the TGF-β superfamily in Drosophila melanogaster. Dev. Biol. 151, 491–505. 1069. Doe, C.Q. (1992). Molecular markers for identiﬁed neuroblasts and ganglion mother cells in the Drosophila central nervous system. Development 116, 855–863. 1070. Doe, C.Q., Chu-LaGraff, Q., Wright, D.M., and Scott, M.P. (1991). The prospero gene speciﬁes cell fates in the Drosophila central nervous system. Cell 65, 451–464. 1071. Doe, C.Q. and Goodman, C.S. (1985). Early events in insect neurogenesis. I. Development and segmental differences in the pattern of neuronal precursor cells. Dev. Biol. 111, 193–205. 1072. Doe, C.Q. and Goodman, C.S. (1985). Early events in insect neurogenesis. II. The role of cell interactions and cell lineage in the determination of neuronal precursor cells. Dev. Biol. 111, 206–219. 1073. Doe, C.Q., Kuwada, J.Y., and Goodman, C.S. (1985). From epithelium to neuroblasts to neurons: the role of cell interactions and cell lineage during insect neurogenesis. Phil. Trans. Roy. Soc. Lond. B 312, 67–81. 1074. Doherty, D., Feger, G., Younger-Shepherd, S., Jan, L.Y., and Jan, Y.N. (1996). Delta is a ventral to dorsal signal complementary to Serrate, another Notch ligand, in Drosophila wing formation. Genes Dev. 10, 421–434. 1075. Doherty, D., Jan, L.Y., and Jan, Y.N. (1997). The Drosophila neurogenic gene big brain, which encodes a membraneassociated protein, acts cell autonomously and can act synergistically with Notch and Delta. Development 124, 3881–3893. 1076. Dokucu, M.E., Zipursky, S.L., and Cagan, R.L. (1996). Atonal, Rough and the resolution of proneural clusters in the developing Drosophila retina. Development 122, 4139–4147. 1077. Dom´ınguez, M. (1999). Dual role for Hedgehog in the regulation of the proneural gene atonal during ommatidia development. Development 126, 2345–2353. 1078. Dom´ınguez, M., Brunner, M., Hafen, E., and Basler, K. (1996). Sending and receiving the Hedgehog signal: control by the Drosophila Gli protein Cubitus interruptus. Science 272, 1621–1625. 1079. Dom´ınguez, M. and Campuzano, S. (1993). asense, a member of the Drosophila achaete-scute complex, is a proneural and neural differentiation gene. EMBO J. 12, 2049– 2060. 1080. Dom´ınguez, M. and de Celis, J.F. (1998). A dorsal/ventral boundary established by Notch controls growth and polarity in the Drosophila eye. Nature 396, 276–278. 1081. Dom´ınguez, M. and Hafen, E. (1996). Genetic dissection of cell fate speciﬁcation in the developing eye of Drosophila. Sems. Cell Dev. Biol. 7, 219–226. 1082. Dom´ınguez, M. and Hafen, E. (1997). Hedgehog directly controls initiation and propagation of retinal differentiation in the Drosophila eye. Genes Dev. 11, 3254– 3264.

REFERENCES

1083. Dom´ınguez, M., Wasserman, J.D., and Freeman, M. (1998). Multiple functions of the EGF receptor in Drosophila eye development. Curr. Biol. 8, 1039–1048. 1084. Dong, C., Li, Z., Alvarez, R., Jr., Feng, X.-H., and Goldschmidt-Clermont, P.J. (2000). Microtubule binding to Smads may regulate TGFβ activity. Molec. Cell 5, 27– 34. 1085. Dong, P.D.S., Chu, J., and Panganiban, G. (2000). Coexpression of the homeobox genes Distal-less and homothorax determines Drosophila antennal identity. Development 127, 209–216. [See also 2001 Development 128, 2365–2372.] 1086. Dong, X., Tsuda, L., Zavitz, K.H., Lin, M., Li, S., Carthew, R.W., and Zipursky, S.L. (1999). ebi regulates epidermal growth factor receptor signaling pathways in Drosophila. Genes Dev. 13, 954–965. 1087. Dong, X., Zavitz, K.H., Thomas, B.J., Lin, M., Campbell, S., and Zipursky, S.L. (1997). Control of G1 in the developing Drosophila eye: rca1 regulates Cyclin A. Genes Dev. 11, 94– 105. 1088. Dooijes, D., van Beest, M., van de Wetering, M., Boulanger, G., Jones, T., Clevers, H., and Mortin, M.A. (1998). Genomic organization of the segment polarity gene pan in Drosophila melanogaster. Mol. Gen. Genet. 258, 45– 52. 1089. Doolittle, R.F. (1995). The multiplicity of domains in proteins. Annu. Rev. Biochem. 64, 287–314. 1090. Doolittle, W.F. (1988). Hierarchical approaches to genome evolution. In “Philosophy and Biology,” (M. Matthen and B. Linsky, eds.). Univ. Calgary Pr.: Calgary, Alberta, Canada, pp. 101–133. 1091. Dorfman, R. and Shilo, B.-Z. (2001). Biphasic activation of the BMP pathway patterns the Drosophila embryonic dorsal region. Development 128, 965–972. 1092. Dorsett, D. (1999). Distant liaisons: long-range enhancerpromoter interactions in Drosophila. Curr. Opin. Gen. Dev. 9, 505–514. 1093. Dougan, S. and DiNardo, S. (1992). Drosophila wingless generates cell type diversity among engrailed expressing cells. Nature 360, 347–350. 1094. Dover, G. (2000). How genomic and developmental dynamics affect evolutionary processes. BioEssays 22, 1153– 1159. 1095. Downing, A.K., Driscoll, P.C., Gout, I., Salim, K., Zvelebil, M.J., and Waterﬁeld, M.D. (1994). Three-dimensional solution structure of the pleckstrin homology domain from dynamin. Curr. Biol. 4, 884–891. 1096. Downward, J. (1995). KSR: a novel player in the RAS pathway. Cell 83, 831–834. 1097. Doyle, D.A., Lee, A., Lewis, J., Kim, E., Sheng, M., and MacKinnon, R. (1996). Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZ. Cell 85, 1067– 1076. 1098. Doyle, H.J. and Bishop, J.M. (1993). Torso, a receptor tyrosine kinase required for embryonic pattern formation, shares substrates with the Sevenless and EGF-R pathways in Drosophila. Genes Dev. 7, 633–646. ˜ 1099. Dreyfuss, G., Matunis, M.J., Pinol-Roma, S., and Burd, C.G. (1993). hnRNP proteins and the biogenesis of mRNA. Annu. Rev. Biochem. 62, 289–321. ˜ 1100. Dreyfuss, G., Swanson, M.S., and Pinol-Roma, S. (1998). Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation. Trends Biochem. Sci. 13, 86–91.

337

1101. Driesch, H. (1894). “Analytische Theorie der organischen Entwicklung.” Engelmann, Leipzig. ¨ 1102. Driever, W. and Nusslein-Volhard, C. (1988). The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner. Cell 54, 95–104. ¨ 1103. Driever, W. and Nusslein-Volhard, C. (1988). A gradient of bicoid protein in Drosophila embryos. Cell 54, 83–93. ¨ 1104. Driever, W., Siegel, V., and Nusslein-Volhard, C. (1990). Autonomous determination of anterior structures in the early Drosophila embryo by the bicoid morphogen. Development 109, 811–820. ¨ 1105. Driever, W., Thoma, G., and Nusslein-Volhard, C. (1989). Determination of spatial domains of zygotic gene expression in the Drosophila embryo by the afﬁnity of binding sites for the bicoid morphogen. Nature 340, 363– 367. ¨ 1106. Dr¨oge, P. and Muller-Hill, B. (2001). High local protein concentrations at promoters: strategies in prokaryotic and eukaryotic cells. BioEssays 23, 179–183. ¨ 1107. Dubendorfer, K. and N¨othiger, R. (1982). A clonal analysis of cell lineage and growth in the male and female genital disc of Drosophila melanogaster. W. Roux’s Arch. 191, 42– 55. 1108. Dubinin, N.P. (1932). Step-allelomorphism and the theory of centres of the gene, achaete-scute. J. Genet. 26, 37–58. 1109. Dubinin, N.P. (1933). Step-allelomorphism in Drosophila melanogaster. J. Genetics 27, 443–464. 1110. Dubnicoff, T., Valentine, S.A., Chen, G., Shi, T., Lengyel, J.A., Paroush, Z., and Courey, A.J. (1997). Conversion of Dorsal from an activator to a repressor by the global corepressor Groucho. Genes Dev. 11, 2952–2957. 1111. Duboule, D. (1994). Temporal colinearity and the phylotypic progression: a basis for the stability of a vertebrate Bauplan and the evolution of morphologies through heterochrony. Development 1994 Suppl., 135–142. 1112. Duboule, D. (1998). Vertebrate Hox gene regulation: clustering and/or colinearity? Curr. Opin. Gen. Dev. 8, 514–518. 1113. Duboule, D. and Morata, G. (1994). Colinearity and functional hierarchy among genes of the homeotic complexes. Trends Genet. 10, 358–364. 1114. Duboule, D. and Wilkins, A.S. (1998). The evolution of “bricolage”. Trends Genet. 14, 54–59. 1115. Dudley, R. (1999). Unsteady aerodynamics. Science 284, 1937–1939. 1116. Dudley, R. (2000). “The Biomechanics of Insect Flight: Form, Function, Evolution.” Princeton Univ. Pr., Princeton, N. J. 1117. Duffy, J.B. and Perrimon, N. (1994). The Torso pathway in Drosophila: lessons on receptor tyrosine kinase signaling and pattern formation. Dev. Biol. 166, 380–395. ˜ 1118. Duggan, A., Garc´ıa-Anoveros, J., and Corey, D.P. (2000). Insect mechanoreception: What a long, strange TRP it’s been. Curr. Biol. 10, R384–R387. 1119. Duncan, D.M., Burgess, E.A., and Duncan, I. (1998). Control of distal antennal identity and tarsal development in Drosophila by spineless-aristapedia, a homolog of the mammalian dioxin receptor. Genes Dev. 12, 1290–1303. 1120. Duncan, I. (1987). The bithorax complex. Annu. Rev. Genet. 21, 285–319. 1121. Duncan, I. (1996). How do single homeotic genes control multiple segment identities? BioEssays 18, 91–94. 1122. Duncan, I.M. (1982). Polycomblike: a gene that appears to be required for the normal expression of the bithorax and antennapedia complexes of Drosophila melanogaster. Genetics 102, 49–70.

338

1123. Dunne, J.F. (1981). Growth dynamics in the regeneration of a fragment of the wing imaginal disc of Drosophila melanogaster. Dev. Biol. 87, 379–382. 1124. Dura, J.-M. and Ingham, P. (1988). Tissue- and stagespeciﬁc control of homeotic and segmentation gene expression in Drosophila embryos by the polyhomeotic gene. Development 103, 733–741. 1125. Duranceau, C., Glenn, S.L., and Schneiderman, H.A. (1980). Positional information as a regulator of growth in the imaginal wing disc of Drosophila melanogaster. In “Insect Biology in the Future,” (M. Locke and D. Smith, eds.). Acad. Pr.: New York, pp. 479–516. 1126. Duronio, R.J. (1999). Establishing links between developmental signaling pathways and cell-cycle regulation in Drosophila. Curr. Opin. Gen. Dev. 9, 81–88. 1127. Dvir, A., Conaway, J.W., and Conaway, R.C. (2001). Mechanism of transcription initiation and promoter escape by RNA polymerase II. Curr. Opin. Gen. Dev. 11, 209–214. 1128. Dye, C.A., Lee, J.-K., Atkinson, R.C., Brewster, R., Han, P.L., and Bellen, H.J. (1998). The Drosophila sanpodo gene controls sibling cell fate and encodes a tropomodulin homolog, an actin/tropomyosin-associated protein. Development 125, 1845–1856. 1129. Dyson, S. and Gurdon, J.B. (1998). The interpretation of position in a morphogen gradient as revealed by occupancy of activin receptors. Cell 93, 557–568. 1130. Easter, S.S., Jr. (2000). Let there be sight. Neuron 27, 193– 195. 1131. Eastman, D.S., Slee, R., Skoufos, E., Bangalore, L., Bray, S., and Delidakis, C. (1997). Synergy between Suppressor of Hairless and Notch in regulation of Enhancer of split mγ and mδ expression. Mol. Cell. Biol. 17, 5620–5628. 1132. Eaton, S. (1997). Planar polarization of Drosophila and vertebrate epithelia. Curr. Opin. Cell Biol. 9, 860–866. 1133. Eaton, S., Auvinen, P., Luo, L., Jan, Y.N., and Simons, K. (1995). CDC42 and Rac1 control different actin-dependent processes in the Drosophila wing disc epithelium. J. Cell Biol. 131, 151–164. 1134. Eaton, S. and Cohen, S. (1996). Wnt signal transduction: more than one way to skin a (β-)cat? Trends Cell Biol. 6, 287–289. 1135. Eaton, S. and Kornberg, T.B. (1990). Repression of ci-D in posterior compartments of Drosophila by engrailed. Genes Dev. 4, 1068–1077. 1136. Eaton, S., Wepf, R., and Simons, K. (1996). Roles for Rac1 and Cdc42 in planar polarization and hair outgrowth in the wing of Drosophila. J. Cell Biol. 135, 1277–1289. 1137. Eberhard, W.G. (2001). Species-speciﬁc genitalic copulatory courtship in sepsid ﬂies (Diptera, Sepsidae, Microsepsis) and theories of genitalic evolution. Evolution 55, 93– 102. 1138. Eberlein, S. and Russell, M.A. (1983). Effects of deﬁciencies in the engrailed region of Drosophila melanogaster. Dev. Biol. 100, 227–237. ¨ T., Nolte, R.T., and Shoel1139. Eck, M.J., Dhe-Paganon, S., Trub, son, S.E. (1996). Structure of the IRS-1 PTB domain bound to the juxtamembrane region of the insulin receptor. Cell 85, 695–705. 1140. Ede, D.A. (1972). Cell behaviour and embryonic development. Internat. J. Neurosci. 3, 165–174. 1141. Edelman, G.M. (1993). Neural Darwinism: selection and reentrant signaling in higher brain function. Neuron 10, 115–125. 1142. Edgar, B.A. and Lehner, C.F. (1996). Developmental control

REFERENCES

1143.

1144.

1145. 1146. 1147.

1148.

1149.

1150.

1151.

1152.

1153.

1154.

1155.

1156.

1157.

1158.

1159.

1160.

of cell cycle regulators: a ﬂy’s perspective. Science 274, 1646–1652. Edgar, B.A., Odell, G.M., and Schubiger, G. (1989). A genetic switch, based on negative regulation, sharpens stripes in Drosophila embryos. Dev. Genet. 10, 124–142. Edlund, T. and Jessell, T.M. (1999). Progression from extrinsic to intrinsic signaling in cell fate speciﬁcation: a view from the nervous system. Cell 96, 211–224. Edwards, J.S. (1994). In memoriam. Sir Vincent Brian Wigglesworth (1899–1994). Dev. Biol. 166, 361–362. Egan, S.E. and Weinberg, R.A. (1993). The pathway to signal achievement. Nature 365, 781–783. Eggert, T., Hauck, B., Hildebrandt, N., Gehring, W.J., and Walldorf, U. (1998). Isolation of a Drosophila homolog of the vertebrate homeobox gene Rx and its possible role in brain and eye development. Proc. Natl. Acad. Sci. USA 95, 2343–2348. Eigen, M. (1971). Selforganization of matter and the evolution of biological macromolecules. Naturwiss. 58, 465– 523. Eisen, J.S. and Youssef, N.N. (1980). Fine structural aspects of the developing compound eye of the honey bee, Apis mellifera L. J. Ultrastr. Res. 71, 79–94. Eisenberg, L.M., Ingham, P.W., and Brown, A.M.C. (1992). Cloning and characterization of a novel Drosophila Wnt gene, Dwnt-5, a putative downstream target of the homeobox gene Distal-less. Dev. Biol. 154, 73–83. Eizinger, A., Jungblut, B., and Sommer, R.J. (1999). Evolutionary change in the functional speciﬁcity of genes. Trends Genet. 15, 197–202. Ellenberger, T., Fass, D., Arnaud, M., and Harrison, S.C. (1994). Crystal structure of transcription factor E47: E-box recognition by a basic region helix-loop-helix dimer. Genes Dev. 8, 970–980. Ellenberger, T.E., Brandl, C.J., Struhl, K., and Harrison, S.C. (1992). The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted α helices: crystal structure of the protein-DNA complex. Cell 71, 1223–1237. Elliott, G. and O’Hare, P. (1997). Intercellular trafﬁcking and protein delivery by a herpesvirus structural protein. Cell 88, 223–233. Ellis, H.M. (1994). Embryonic expression and function of the Drosophila helix-loop-helix gene, extramacrochaetae. Mechs. Dev. 47, 65–72. Ellis, H.M., Spann, D.R., and Posakony, J.W. (1990). extramacrochaetae, a negative regulator of sensory organ development in Drosophila, deﬁnes a new class of helixloop-helix proteins. Cell 61, 27–38. Ellis, M.C., O’Neill, E.M., and Rubin, G.M. (1993). Expression of Drosophila glass protein and evidence for negative regulation of its activity in non-neuronal cells by another DNA-binding protein. Development 119, 855–865. Ellis, M.C., Weber, U., Wiersdorff, V., and Mlodzik, M. (1994). Confrontation of scabrous expressing and nonexpressing cells is essential for normal ommatidial spacing in the Drosophila eye. Development 120, 1959–1969. [Cf. 2001 Dev. Biol. 240, 361–376.] Elsdale, T. and Wasoff, F. (1976). Fibroblast cultures and dermatoglyphics: the topology of two planar patterns. W. Roux’s Arch. 180, 121–147. Elsholtz, H.P., Albert, V.R., Treacy, M.N., and Rosenfeld, M.G. (1990). A two-base change in a POU factor-binding site switches pituitary-speciﬁc to lymphoid-speciﬁc gene expression. Genes Dev. 4, 43–51.

REFERENCES

1161. Elstob, P.R., Brodu, V., and Gould, A.P. (2001). spaltdependent switching between two cell fates that are induced by the Drosophila EGF receptor. Development 128, 723–732. 1162. Emerald, B.S. and Roy, J.K. (1997). Homeotic transformation in Drosophila. Nature 389, 684. 1163. Emerald, B.S. and Roy, J.K. (1998). Organising activities of engrailed, hedgehog, wingless and decapentaplegic in the genital discs of Drosophila melanogaster. Dev. Genes Evol. 208, 504–516. 1164. Emery, J.F. and Bier, E. (1995). Speciﬁcity of CNS and PNS regulatory subelements comprising pan-neural enhancers of the deadpan and scratch genes is achieved by repression. Development 121, 3549–3560. 1165. Emlen, D.J. and Nijhout, H.F. (2000). The development and evolution of exaggerated morphologies in insects. Annu. Rev. Entomol. 45, 661–708. 1166. Emmons, R.B., Duncan, D., Estes, P.A., Kiefel, P., Mosher, J.T., Sonnenfeld, M., Ward, M.P., Duncan, I., and Crews, S.T. (1999). The Spineless-Aristapedia and Tango bHLHPAS proteins interact to control antennal and tarsal development in Drosophila. Development 126, 3937–3945. 1167. Engstrom, L., Noll, E., and Perrimon, N. (1997). Paradigms to study signal transduction pathways in Drosophila. Curr. Topics Dev. Biol. 35, 229–261. 1168. Engstr¨om, Y., Schneuwly, S., and Gehring, W.J. (1992). Spatial and temporal expression of an Antennapedia/lac Z gene construct integrated into the endogenous Antennapedia gene of Drosophila melanogaster. Roux’s Arch. Dev. Biol. 201, 65–80. 1169. Entchev, E.V., Schwabedissen, A., and Gonz´alez-Gait´an, M. (2000). Gradient formation of the TGF-β homolog Dpp. Cell 103, 981–991. 1170. Epper, F. and N¨othiger, R. (1982). Genetic and developmental evidence for a repressed genital primordium in Drosophila melanogaster. Dev. Biol. 94, 163–175. 1171. Epps, J.L., Jones, J.B., and Tanda, S. (1997). oroshigane, a new segment polarity gene of Drosophila melanogaster, functions in Hedgehog signal transduction. Genetics 145, 1041–1052. 1172. Epstein, E.A., Martin, J.L., Haerry, T., Lincecum, J., and O’Connor, M.B. (2001). Genetic investigation of the sara and syndecan region. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a145. 1173. Erickson, J.W. and Cline, T.W. (1993). A bZIP protein, Sisterless-a, collaborates with bHLH transcription factors early in Drosophila development to determine sex. Genes Dev. 7, 1688–1702. 1174. Erickson, J.W. and Cline, T.W. (1998). Key aspects of the primary sex determination mechanism are conserved across the genus Drosophila. Development 125, 3259–3268. 1175. Erkner, A., Gallet, A., Angelats, C., Fasano, L., and Kerridge, S. (1999). The role of Teashirt in proximal leg development in Drosophila: ectopic teashirt expression reveals different cell behaviours in ventral and dorsal domains. Dev. Biol. 215, 221–232. 1176. Erneux, T., Hiernaux, J., and Nicolis, G. (1978). Turing’s theory in morphogenesis. Bull. Math. Biol. 40, 771– 789. 1177. Erwin, D.H. and Valentine, J.W. (1984). “Hopeful monsters,” transposons, and metazoan radiation. Proc. Natl. Acad. Sci. USA 81, 5482–5483. 1178. Eskens, A.A.C., Sprey, T.E., and Westra, A. (1981). Morphological indications for compartmental boundaries in the

339

1179.

1180.

1181. 1182.

1183.

1184.

1185.

1186.

1187.

1188.

1189.

1190.

1191.

1192.

1193.

1194.

1195.

imaginal wing disc of Drosophila melanogaster. Neth. J. Zool. 31, 773–776. Estrada, B. and S´anchez-Herrero, E. (2001). The Hox gene Abdominal-B antagonizes appendage development in the genital disc of Drosophila. Development 128, 331–339. ¨ Fagotto, F., Gluck, U., and Gumbiner, B.M. (1998). Nuclear localization signal-independent and importin/ karyopherin-independent nuclear import of β-catenin. Curr. Biol. 8, 181–190. Fagotto, F. and Gumbiner, B.M. (1996). Cell contactdependent signaling. Dev. Biol. 180, 445–454. Fagotto, F., Jho, E.-h., Zeng, L., Kurth, T., Joos, T., Kaufmann, C., and Costantini, F. (1999). Domains of Axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization. J. Cell Biol. 145, 741–756. Fain, M.J. and Schneiderman, H.A. (1979). Regulative interaction of mature imaginal disc fragments with embryonic and immature disc tissues in Drosophila. W. Roux’s Arch. 187, 1–11. Fain, M.J. and Schneiderman, H.A. (1979). Wound healing and regenerative response of fragments of the Drosophila wing imaginal disc cultured in vitro. J. Insect Physiol. 25, 913–924. Fain, M.J. and Stevens, B. (1982). Alterations in the cell cycle of Drosophila imaginal disc cells precede metamorphosis. Dev. Biol. 92, 247–258. Fairall, L., Harrison, S.D., Travers, A.A., and Rhodes, D. (1992). Sequence-speciﬁc DNA binding by a two zincﬁnger peptide from the Drosophila melanogaster Tramtrack protein. J. Mol. Biol. 226, 349–366. Fairall, L., Schwabe, J.W.R., Chapman, L., Finch, J.T., and Rhodes, D. (1993). The crystal structure of a two zincﬁnger peptide reveals an extension to the rules for zincﬁnger/DNA recognition. Nature 366, 483–487. Falvo, J.W., Thanos, D., and Maniatis, T. (1995). Reversal of intrinsic DNA bends in the IFNb gene enhancer by transcription factors and the architectural protein HMG I(Y). Cell 83, 1101–1111. Fan, X., Brass, L.F., Poncz, M., Spitz, F., Maire, P., and Manning, D.R. (2000). The a subunits of Gz and Gi interact with the eyes absent transcription cofactor Eya2, preventing its interaction with the Six class of homeodomain-containing proteins. J. Biol. Chem. 275 #41, 32129–32134. Fankhauser, G. (1945). The effects of changes in chromosome number on amphibian development. Q. Rev. Biol. 20, 20–78. Fankhauser, G. (1955). The role of nucleus and cytoplasm. In “Analysis of Development,” (B.H. Willier, P.A. Weiss, and V. Hamburger, eds.). Hafner: New York, pp. 126–150. Fanning, A.S. and Anderson, J.M. (1996). Protein-protein interactions: PDZ domain networks. Curr. Biol. 6, 1385– 1388. Fanto, M., Mayes, C.A., and Mlodzik, M. (1998). Linking cell-fate speciﬁcation to planar polarity: determination of the R3/R4 photoreceptors is a prerequisite for the interpretation of the Frizzled mediated polarity signal. Mechs. Dev. 74, 51–58. Fanto, M. and Mlodzik, M. (1999). Asymmetric Notch activation speciﬁes photoreceptors R3 and R4 and planar polarity in the Drosophila eye. Nature 397, 523–526. Fanto, M., Weber, U., Strutt, D.I., and Mlodzik, M. (2000). Nuclear signaling by Rac and Rho GTPases is required

340

1196.

1197.

1198.

1199.

1200. 1201.

1202.

1203.

1204.

1205.

1206.

1207.

1208.

1209.

1210.

1211.

1212.

REFERENCES

in the establishment of epithelial planar polarity in the Drosophila eye. Curr. Biol. 10, 979–988. Farrar, M.A., Alberola-Ila, J., and Perlmutter, R.M. (1996). Activation of the Raf-1 kinase cascade by coumermycininduced dimerization. Nature 383, 178–181. Fasano, L., R¨oder, L., Cor´e, N., Alexandre, E., Vola, C., Jacq, B., and Kerridge, S. (1991). The gene teashirt is required for the development of Drosophila embryonic trunk segments and encodes a protein with widely spaced zinc ﬁnger motifs. Cell 64, 63–79. Fausto-Sterling, A. and Hsieh, L. (1978). Pattern formation in the wing veins of the fused mutant (Drosophila melanogaster). Dev. Biol. 63, 358–369. Fauvarque, M.-O. and Dura, J.-M. (1993). polyhomeotic regulatory sequences induce developmental regulatordependent variegation and targeted P-element insertions in Drosophila. Genes Dev. 7, 1508–1520. Featherstone, C. (1997). Src structure crystallizes 20 years of oncogene research. Science 275, 1066. Feger, G., Vaessin, H., Su, T.T., Wolff, E., Jan, L.Y., and Jan, Y.N. (1995). dpa, a member of the MCM family, is required for mitotic DNA replication but not endoreplication in Drosophila. EMBO J. 14, 5387–5398. Fehon, R.G., Gauger, A., and Schubiger, G. (1987). Cellular recognition and adhesion in embryos and imaginal discs of Drosophila melanogaster. In “Genetic Regulation of Development,” Symp. Soc. Dev. Biol., Vol. 45 (W.F. Loomis, ed.). Alan R. Liss: New York, pp. 141–170. Fehon, R.G., Johansen, K., Rebay, I., and ArtavanisTsakonas, S. (1991). Complex cellular and subcellular regulation of Notch expression during embryonic and imaginal development of Drosophila: Implications for Notch function. J. Cell Biol. 113, 657–669. Fehon, R.G., Kooh, P.J., Rebay, I., Regan, C.L., Xu, T., Muskavitch, M.A.T., and Artavanis-Tsakonas, S. (1990). Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGFhomologous genes in Drosophila. Cell 61, 523–534. Fehon, R.G. and Schubiger, G. (1985). Dissociation and sorting out of Drosophila imaginal disc cells. Dev. Biol. 108, 465–473. Feinbaum, R. and Ambros, V. (1999). The timing of lin4 RNA accumulation controls the timing of postembryonic developmental events in Caenorhabditis elegans. Dev. Biol. 210, 87–95. Feldman, P., Eicher, E.N., Leevers, S.J., Hafen, E., and Hughes, D.A. (1999). Control of growth and differentiation by Drosophila RasGAP, a homolog of p120 Ras-GTPaseactivating protein. Mol. Cell. Biol. 19, 1928–1937. Felsenfeld, A.L. and Kennison, J.A. (1995). Positional signaling by hedgehog in Drosophila imaginal disc development. Development 121, 1–10. Feng, G.-S. and Pawson, T. (1994). Phosphotyrosine phosphatases with SH2 domains: regulators of signal transduction. Trends Genet. 10, 54–58. Feng, S., Chen, J.K., Yu, H., Simon, J.A., and Schreiber, S.L. (1994). Two binding orientations for peptides to the Src SH3 domain: development of a general model for SH3ligand interactions. Science 266, 1241–1247. Ferguson, E.L. and Anderson, K.V. (1992). decapentaplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophila embryo. Cell 71, 451–461. Ferguson, E.L. and Anderson, K.V. (1992). Localized enhancement and repression of the activity of the TGF-β

1213.

1214.

1215.

1216.

1217.

1218.

1219.

1220.

1221.

1222.

1223. 1224.

1225.

1226.

1227. 1228. 1229.

1230.

1231.

family member, decapentaplegic, is necessary for dorsalventral pattern formation in the Drosophila embryo. Development 114, 583–597. Ferguson, K.M., Kavran, J.M., Sankaran, V.G., Fournier, E., Isakoff, S.J., Skolnik, E.Y., and Lemmon, M.A. (2000). Structural basis for discrimination of 3-phosphoinositides by pleckstrin homology domains. Molec. Cell 6, 373–384. Fernandes, J., Celniker, S.E., Lewis, E.B., and VijayRaghavan, K. (1994). Muscle development in the four-winged Drosophila and the role of the Ultrabithorax gene. Curr. Biol. 4, 957–964. Fernandez, R., Tabarini, D., Azpiazu, N., Frasch, M., and Schlessinger, J. (1995). The Drosophila insulin receptor homolog: a gene essential for embryonic development encodes two receptor isoforms with different signaling potential. EMBO J. 14, 3373–3384. Fernandez-Almonacid, R. and Rosen, O.M. (1987). Structure and ligand speciﬁcity of the Drosophila melanogaster insulin receptor. Mol. Cell. Biol. 7, 2718–2727. ´ Fern´andez-Funez, P., Lu, C.-H., Rinc´on-Limas, D.E., Garc´ıa-Bellido, A., and Botas, J. (1998). The relative expression amounts of apterous and its co-factor dLdb/Chip are critical for dorso-ventral compartmentalization in the Drosophila wing. EMBO J. 17, 6846–6853. Fernandez-Funez, P., Rickhof, G., Morata, G., and Botas, J. (2001). dlim1 plays an essential role in the speciﬁcation of ventral appendages. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a198. ¨ Ferrandon, D., Koch, I., Westhof, E., and Nusslein-Volhard, C. (1997). RNA-RNA interaction is required for the formation of speciﬁc bicoid mRNA 3 UTR-STAUFEN ribonucleoprotein particles. EMBO J. 16, 1751–1758. Ferrari, S., Harley, V.R., Pontiggia, A., Goodfellow, P.N., Lovell-Badge, R., and Bianchi, M.E. (1992). SRY, like HMG1, recognizes sharp angles in DNA. EMBO J. 11, 4497–4506. Ferr´e-D’Amar´e, A.R., Pognonec, P., Roeder, R.G., and Burley, S.K. (1994). Structure and function of the b/HLH/Z domain of USF. EMBO J. 13, 180–189. Ferrell, J.E., Jr. and Machleder, E.M. (1998). The biochemical basis of an all-or-none cell fate switch in Xenopus oocytes. Science 280, 895–898. Ferrier, D.E.K. and Holland, P.W.H. (2001). Ancient origin of the Hox gene cluster. Nature Rev. Gen. 2, 33–38. Ferris, G.F. (1950). External morphology of the adult. In “Biology of Drosophila,” (M. Demerec, ed.). Hafner: New York, pp. 368–419. Ferrus, A. (1979). Cell functions in morphogenesis: clonal analysis of new morphogenetic mutations in Drosophila melanogaster. Dev. Biol. 68, 16–28. ´ A. and Garcia-Bellido, A. (1976). Morphogenetic Ferrus, mutants detected in mitotic recombination clones. Nature 260, 425–426. ffrench-Constant, C. (1994). How do embryonic cells measure time? Curr. Biol. 4, 415–419. Fields, S. and Song, O.-k. (1989). A novel genetic system to detect protein-protein interactions. Nature 340, 245–246. Fields, S. and Sternglanz, R. (1994). The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 10, 286–292. Fiering, S., Whitelaw, E., and Martin, D.I.K. (2000). To be or not to be active: the stochastic nature of enhancer action. BioEssays 22, 381–387. Fietz, M.J., Jacinto, A., Taylor, A.M., Alexandre, C., and

REFERENCES

1232.

1233.

1234. 1235.

1236.

1237.

1238.

1239.

1240.

1241.

1242.

1243.

1244.

1245.

1246.

1247.

Ingham, P.W. (1995). Secretion of the amino-terminal fragment of the Hedgehog protein is necessary and sufﬁcient for hedgehog signalling in Drosophila. Curr. Biol. 6, 643–650. Finelli, A.L., Bossie, C.A., Xie, T., and Padgett, R.W. (1994). Mutational analysis of the Drosophila tolloid gene, a human BMP-1 homologue. Development 120, 861–870. Finelli, A.L., Xie, T., Bossie, C.A., Blackman, R.K., and Padgett, R.W. (1995). The tolkin gene is a tolloid/BMP-1 homologue that is essential for Drosophila development. Genetics 141, 271–281. Fini, M.E., Strissel, K.J., and West-Mays, J.A. (1997). Perspectives on eye development. Dev. Genet. 20, 175–185. Finkbohner, J.D., Cunningham, G.N., and Kuhn, D.T. (1985). Tissue distribution of malic enzyme-NADP+ in D. melanogaster imaginal discs. Roux’s Arch. Dev. Biol. 194, 217–223. Finnerty, J.R. and Martindale, M.Q. (1998). The evolution of the Hox cluster: insights from outgroups. Curr. Opin. Gen. Dev. 8, 681–687. [See also 2001 Curr. Biol. 11, 759– 763.] Firth, L., Manchester, J., Lorenzen, J.A., Baron, M., and Perkins, L.A. (2000). Identiﬁcation of genomic regions that interact with a viable allele of the Drosophila protein tyrosine phosphatase Corkscrew. Genetics 156, 733–748. Fischer, J.A., Leavell, S.K., and Li, Q. (1997). Mutagenesis screens for interacting genes reveal three roles for fat facets during Drosophila eye development. Dev. Genet. 21, 167– 174. Fischer-Vize, J.A. and Mosley, K.L. (1994). marbles mutants: uncoupling cell determination and nuclear migration in the developing Drosophila eye. Development 120, 2609–2618. Fischer-Vize, J.A., Rubin, G.M., and Lehmann, R. (1992). The fat facets gene is required for Drosophila eye and embryo development. Development 116, 985–1000. Fischer-Vize, J.A., Vize, P.D., and Rubin, G.M. (1992). A unique mutation in the Enhancer of split gene complex affects the fates of the mystery cells in the developing Drosophila eye. Development 115, 89–101. Fisher, A. and Caudy, M. (1998). The function of hairyrelated bHLH repressor proteins in cell fate decisions. BioEssays 20, 298–306. Fisher, A.L. and Caudy, M. (1998). Groucho proteins: transcriptional corepressors for speciﬁc subsets of DNAbinding transcription factors in vertebrates and invertebrates. Genes Dev. 12, 1931–1940. Fisher, A.L., Ohsako, S., and Caudy, M. (1996). The WRPW motif of the Hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain. Mol. Cell. Biol. 16, 2670–2677. Fisher, F. and Goding, C.R. (1992). Single amino acid substitutions alter helix-loop-helix protein speciﬁcity for bases ﬂanking the core CANNTG motif. EMBO J. 11, 4103–4109. Fitzpatrick, C.A., Sharkov, N., and Katzen, A.L. (2001). Drosophila myb may play a role in regulating differentiation as well as proliferation during development. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a64. Fjose, A., McGinnis, W.J., and Gehring, W.J. (1985). Isolation of a homoeo box-containing gene from the engrailed region of Drosophila and the spatial distribution of its transcripts. Nature 313, 284–289.

341

1248. Flegel, W.A., Singson, A.W., Margolis, J.S., Bang, A.G., Posakony, J.W., and Murre, C. (1993). Dpbx, a new homeobox gene closely related to the human proto-oncogene pbx1: molecular structure and developmental expression. Mechs. Dev. 41, 155–161. 1249. Fleming, R.J. (1998). Structural conservation of Notch receptors and ligands. Sems. Cell Dev. Biol. 9, 599–607. 1250. Fleming, R.J., Gu, Y., and Hukriede, N.A. (1997). Serratemediated activation of Notch is speciﬁcally blocked by the product of the gene fringe in the dorsal compartment of the Drosophila wing imaginal disc. Development 124, 2973–2981. 1251. Fleming, R.J., Purcell, K., and Artavanis-Tsakonas, S. (1997). The NOTCH receptor and its ligands. Trends Cell Biol. 7, 437–441. 1252. Fleming, R.J., Scottgale, T.N., Diederich, R.J., and Artavanis-Tsakonas, S. (1990). The gene Serrate encodes a putative EGF-like transmembrane protein essential for proper ectodermal development in Drosophila melanogaster. Genes Dev. 4, 2188–2201. 1253. Flenniken, A.M., Gale, N.W., Yancopoulos, G.D., and Wilkinson, D.G. (1996). Distinct and overlapping expression patterns of ligands for Eph-related receptor tyrosine kinases during mouse embryogenesis. Dev. Biol. 179, 382– 401. 1254. Fletcher, J.C. and Thummel, C.S. (1995). The ecdysoneinducible Broad-Complex and E 74 early genes interact to regulate target gene transcription and Drosophila metamorphosis. Genetics 141, 1025–1035. 1255. Flores, G.V., Daga, A., Kalhor, H.R., and Banerjee, U. (1998). Lozenge is expressed in pluripotent precursor cells and patterns multiple cell types in the Drosophila eye through the control of cell-speciﬁc transcription factors. Development 125, 3681–3687. 1256. Flores, G.V., Duan, H., Yan, H., Nagaraj, R., Fu, W., Zou, Y., Noll, M., and Banerjee, U. (2000). Combinatorial signaling in the speciﬁcation of unique cell fates. Cell 103, 75–85. 1257. Flores-Saaib, R.D., Jia, S., and Courey, A.J. (2001). Activation and repression by the C-terminal domain of Dorsal. Development 128, 1869–1879. 1258. Foe, V.E. (1989). Mitotic domains reveal early commitment of cells in Drosophila embryos. Development 107, 1–22. 1259. Foe, V.E., Odell, G.M., and Edgar, B.A. (1993). Mitosis and morphogenesis in the Drosophila embryo: point and counterpoint. In “The Development of Drosophila melanogaster,” Vol. 1 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Lab. Pr.: Plainview, N. Y., pp. 149–300. 1260. Foelix, R.F., Stocker, R.F., and Steinbrecht, R.A. (1989). Fine structure of a sensory organ in the arista of Drosophila melanogaster and some other dipterans. Cell Tissue Res. 258, 277–287. 1261. Follette, P.J., Duronio, R.J., and O’Farrell, P.H. (1998). Fluctuations in Cyclin E levels are required for multiple rounds of endocycle S phase in Drosophila. Curr. Biol. 8, 235–238. 1262. Follette, P.J. and O’Farrell, P.H. (1997). Cdks and the Drosophila cell cycle. Curr. Opin. Gen. Dev. 7, 17–22. 1263. Follette, P.J. and O’Farrell, P.H. (1997). Connecting cell behavior to patterning: lessons from the cell cycle. Cell 88, 309–314. 1264. Forbes, A.J., Nakano, Y., Taylor, A.M., and Ingham, P.W. (1993). Genetic analysis of hedgehog signalling in the Drosophila embryo. Development 1993 Suppl., 115–124. 1265. Force, A., Lynch, M., Pickett, F.B., Amores, A., Yan, Y.-l., and Postlethwait, J. (1999). Preservation of duplicate genes

342

1266. 1267.

1268. 1269.

1270.

1271.

1272.

1273.

1274.

1275.

1276.

1277.

1278.

1279.

1280.

1281.

1282. 1283.

1284. 1285.

REFERENCES

by complementary, degenerative mutations. Genetics 151, 1531–1545. Ford, B.J. (1998). The earliest views. Sci. Am. 278 #4, 50–53. Fornerod, M., Ohno, M., Yoshida, M., and Mattaj, I.W. (1997). CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90, 1051–1060. Fortini, M. (2000). Fringe beneﬁts to carbohydrates. Nature 406, 357–358. Fortini, M.E. and Artavanis-Tsakonas, S. (1994). The Suppressor of Hairless protein participates in Notch receptor signaling. Cell 79, 273–282. Fortini, M.E. and Bonini, N.M. (2000). Modeling human neurodegenerative diseases in Drosophila. Trends Genet. 16, 161–167. Fortini, M.E., Rebay, I., Caron, L.A., and ArtavanisTsakonas, S. (1993). An activated Notch receptor blocks cell-fate commitment in the developing Drosophila eye. Nature 365, 555–557. Fortini, M.E. and Rubin, G.M. (1990). Analysis of cis-acting requirements of the Rh3 and Rh4 genes reveals a bipartite organization to the rhodopsin promoters in Drosophila melanogaster. Genes Dev. 4, 444–463. Fortini, M.E., Simon, M.A., and Rubin, G.M. (1992). Signalling by the sevenless protein tyrosine kinase is mimicked by Ras1 activation. Nature 355, 559–561. Foster, G.G. (1975). Negative complementation at the Notch locus of Drosophila melanogaster. Genetics 81, 99– 120. Fostier, M. and Baron, M. (2001). Suppressor of deltex needs deltex to down-regulate the Notch pathway during wing development. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a34. Fostier, M., Evans, D.A.P., Artavanis-Tsakonas, S., and Baron, M. (1998). Genetic characterization of the Drosophila melanogaster Suppressor of deltex gene: a regulator of Notch signaling. Genetics 150, 1477–1485. Fradkin, L.G., Noordermeer, J.N., and Nusse, R. (1995). The Drosophila Wnt protein DWnt-3 is a secreted glycoprotein localized on the axon tracts of the embryonic CNS. Dev. Biol. 168, 202–213. Franc¸ois, V., Solloway, M., O’Neill, J.W., Emery, J., and Bier, E. (1994). Dorsal-ventral patterning of the Drosophila embryo depends on a putative negative growth factor encoded by the short gastrulation gene. Genes Dev. 8, 2602– 2616. Frankfort, B.J., Nolo, R., Zhang, Z., Bellen, H.J., and Mardon, G. (2001). senseless repression of rough is required for R8 photoreceptor differentiation in the developing Drosophila eye. Neuron 32, 403–414. Franks, R.G. and Crews, S.T. (1994). Transcriptional activation domains of the single-minded bHLH protein are required for CNS midline cell development. Mechs. Dev. 45, 269–277. Frasch, M. (1995). Induction of visceral and cardiac mesoderm by ectodermal Dpp in the early Drosophila embryo. Nature 374, 464–467. Fraser, A. (1963). Variation of scutellar bristles in Drosophila. I. Genetic leakage. Genetics 48, 497–514. Fraser, A.G., McCarthy, N.J., and Evan, G.I. (1997). drICE is an essential caspase required for apoptotic activity in Drosophila cells. EMBO J. 16, 6192–6199. Fraser, S.E. and Harland, R.M. (2000). The molecular metamorphosis of experimental embryology. Cell 100, 41–55. Frayne, E.G. and Sato, T. (1991). The Ultrabithorax gene

1286. 1287.

1288.

1289.

1290.

1291. 1292.

1293.

1294.

1295.

1296.

1297.

1298. 1299. 1300.

1301.

1302.

1303. 1304. 1305.

1306.

of Drosophila and the speciﬁcation of abdominal histoblasts. Dev. Biol. 146, 265–277. Freeman, M. (1991). First, trap your enhancer. Curr. Biol. 1, 378–381. Freeman, M. (1994). Misexpression of the Drosophila argos gene, a secreted regulator of cell determination. Development 120, 2297–2304. Freeman, M. (1994). The spitz gene is required for photoreceptor determination in the Drosophila eye where it interacts with the EGF receptor. Mechs. Dev. 48, 25–33. Freeman, M. (1996). Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye. Cell 87, 651–660. Freeman, M. (1997). Cell determination strategies in the Drosophila eye. Development 124, 261–270. [See also 2001 Mechs. Dev. 108, 13–27.] Freeman, M. (1998). Complexity of EGF receptor signalling revealed in Drosophila. Curr. Opin. Gen. Dev. 8, 407–411. Freeman, M. (2000). Feedback control of intercellular signalling in development. Nature 408, 313–319. [See also 2001 EMBO J. 20, 2528–2535.] Freeman, M., Kimmel, B.E., and Rubin, G.M. (1992). Identifying targets of the rough homeobox gene of Drosophila: evidence that rhomboid functions in eye development. Development 116, 335–346. Freeman, M., Kl¨ambt, C., Goodman, C.S., and Rubin, G.M. (1992). The argos gene encodes a diffusible factor that regulates cell fate decisions in the Drosophila eye. Cell 69, 963–975. Freemont, P.S. (1993). The RING ﬁnger: a novel protein sequence motif related to the zinc ﬁnger. Annals N. Y. Acad. Sci. 684, 174–192. Freigang, J., Proba, K., Leder, L., Diederichs, K., Sonderegger, P., and Welte, W. (2000). The crystal structure of the ligand binding module of Axonin-1/TAG-1 suggests a zipper mechanism for neural cell adhesion. Cell 101, 425–433. French, V. (1980). Positional information around the segments of the cockroach leg. J. Embryol. Exp. Morph. 59, 281–313. French, V. (1981). Pattern regulation and regeneration. Phil. Trans. Roy. Soc. Lond. B 295, 601–617. French, V. (1982). Leg regeneration in insects: cell interactions and lineage. Amer. Zool. 22, 79–90. French, V. (1983). Development and evolution of the insect segment. In “Development and Evolution,” Symp. Brit. Soc. Dev. Biol., Vol. 6 (B.C. Goodwin, N. Holder, and C.C. Wylie, eds.). Cambridge Univ. Pr.: Cambridge, pp. 161–193. French, V. (1984). A model of insect limb regeneration. In “Pattern Formation: A Primer in Developmental Biology,” (G.M. Malacinski and S.V. Bryant, eds.). Macmillan: New York, pp. 339–364. French, V. (1984). The structure of supernumerary leg regenerates in the cricket. J. Embryol. Exp. Morph. 81, 185– 209. French, V., Bryant, P.J., and Bryant, S.V. (1976). Pattern regulation in epimorphic ﬁelds. Science 193, 969–981. French, V. and Daniels, G. (1994). The beginning and the end of insect limbs. Curr. Biol. 4, 34–37. Friedrich, M., Rambold, I., and Melzer, R.R. (1996). The early stages of ommatidial development in the ﬂour beetle Tribolium castaneum (Coleoptera; Tenebrionidae). Dev. Genes Evol. 206, 136–146. Frigerio, G., Burri, M., Bopp, D., Baumgartner, S., and Noll, M. (1986). Structure of the segmentation gene paired and

REFERENCES

1307.

1308. 1309.

1310.

1311.

1312.

1313.

1314. 1315.

1316. 1317. 1318.

1319.

1320.

1321.

1322.

1323.

1324.

the Drosophila PRD gene set as part of a gene network. Cell 47, 735–746. Frise, E., Knoblich, J.A., Younger-Shepherd, S., Jan, L.Y., and Jan, Y.N. (1996). The Drosophila Numb protein inhibits signaling of the Notch receptor during cell-cell interaction in sensory organ lineage. Proc. Natl. Acad. Sci. USA 93, 11925–11932. Fris´en, J. and Lendahl, U. (2001). Oh no, Notch again! BioEssays 23, 3–7. Fristrom, D. (1969). Cellular degeneration in the production of some mutant phenotypes in Drosophila melanogaster. Mol. Gen. Genet. 103, 363–379. Fristrom, D. (1972). Chemical modiﬁcation of cell death in the Bar eye of Drosophila. Molec. Gen. Genet. 115, 10– 18. Fristrom, D. and Fristrom, J.W. (1993). The metamorphic development of the adult epidermis. In “The Development of Drosophila melanogaster,” Vol. 2 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Lab. Pr.: Plainview, N. Y., pp. 843–897. Fristrom, D., Gotwals, P., Eaton, S., Kornberg, T.B., Sturtevant, M., Bier, E., and Fristrom, J. (1994). blistered: a gene required for vein/intervein formation in wings of Drosophila. Development 120, 2661–2671. Fristrom, D., Wilcox, M., and Fristrom, J. (1993). The distribution of PS integrins, laminin A and F-actin during key stages in Drosophila wing development. Development 117, 509–523. Fristrom, J.W. (1970). The developmental biology of Drosophila. Annu. Rev. Genet. 4, 325–346. Fristrom, J.W. (1972). The biochemistry of imaginal disk development. In “The Biology of Imaginal Disks,” Results and Problems in Cell Differentiation, Vol. 5, (H. Ursprung and R. N¨othiger, eds.). Springer-Verlag: Berlin, pp. 109– 154. Fritz, C.C. and Green, M.R. (1992). Fishing for partners. Curr. Biol. 2, 403–405. Fry, C.J. and Peterson, C.L. (2001). Chromatin remodeling enzymes: who’s on ﬁrst? Curr. Biol. 11, R185–R197. Fryxell, K.J. and Wood, C.P. (1995). Genetic mosaic analysis of the equatorial-less mutation in Drosophila melanogaster. Dev. Genet. 16, 264–272. Fu, W., Duan, H., Frei, E., and Noll, M. (1998). shaven and sparkling are mutations in separate enhancers of the Drosophila Pax2 homolog. Development 125, 2943–2950. Fu, W. and Noll, M. (1997). The Pax2 homolog sparkling is required for development of cone and pigment cells in the Drosophila eye. Genes Dev. 11, 2066–2078. Fuerstenberg, S., Broadus, J., and Doe, C.Q. (1998). Asymmetry and cell fate in the Drosophila embryonic CNS. Int. J. Dev. Biol. 42, 379–383. Fuerstenberg, S., Peng, C.-Y., Alvarez-Ortiz, P., Hor, T., and Doe, C.Q. (1998). Identiﬁcation of Miranda protein domains regulating asymmetric cortical localization, cargo binding, and cortical release. Molec. Cell. Neurosci. 12, 325–339. Fujioka, M., Emi-Sarker, Y., Yusibova, G.L., Goto, T., and Jaynes, J.B. (1999). Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients. Development 126, 2527–2538. Fujioka, M., Jaynes, J.B., and Goto, T. (1995). Early evenskipped stripes act as morphogenetic gradients at the sin-

343

1325.

1326.

1327.

1328.

1329.

1330.

1331.

1332.

1333.

1334.

1335.

1336.

1337.

1338.

1339.

1340.

1341.

gle cell level to establish engrailed expression. Development 121, 4371–4382. Fujioka, M., Miskiewicz, P., Raj, L., Gulledge, A.A., Weir, M., and Goto, T. (1996). Drosophila Paired regulates late evenskipped expression through a composite binding site for the paired domain and the homeodomain. Development 122, 2697–2707. Fuks, F., Burgers, W.A., Brehm, A., Hughes-Davies, L., and Kouzarides, T. (2000). DNA methyltransferase Dnmt1 associates with histone deacetylase activity. Nature Genet. 24, 88–91. Funakoshi, Y., Minami, M., and Tabata, T. (2001). mtv shapes the activity gradient of the Dpp morphogen through regulation of thickveins. Development 128, 67– 74. Furman, D.P., Rodin, S.N., and Ratner, V.A. (1979). Structural and functional analysis of the scute locus in Drosophila melanogaster. Theor. Appl. Genet. 55, 231– 238. Furriols, M. and Bray, S. (2000). Dissecting the mechanisms of Suppressor of Hairless function. Dev. Biol. 227, 520–532. Furriols, M. and Bray, S. (2001). A model Notch response element detects Suppressor of Hairless-dependent molecular switch. Curr. Biol. 11, 60–64. Furukawa, T., Kawaichi, M., Matsunami, N., Ryo, H., Nishida, Y., and Honjo, T. (1991). The Drosophila RBP-Jκ gene encodes the binding protein for the immunoglobulin Jκ recombination signal sequence. J. Biol. Chem. 266 #34, 23334–23340. Furukawa, T., Kobayakawa, Y., Tamura, K., Kimura, K.-I., Kawaichi, M., Tanimura, T., and Honjo, T. (1995). Suppressor of Hairless, the Drosophila homologue of RBP-Jκ, transactivates the neurogenic gene E(spl)m8. Jpn. J. Genet. 70, 505–524. Fuse, N., Hirose, S., and Hayashi, S. (1996). Determination of wing cell fate by the escargot and snail genes in Drosophila. Development 122, 1059–1067. Fuse, N., Matakatsu, H., Taniguchi, M., and Hayashi, S. (1999). Snail-type zinc ﬁnger proteins prevent neurogenesis in Scutoid and transgenic animals of Drosophila. Dev. Genes Evol. 209, 573–580. Fuss, B. and Hoch, M. (1998). Drosophila endoderm development requires a novel homeobox gene which is a target of Wingless and Dpp signalling. Mechs. Dev. 79, 83–97. Fuss, B., Meissner, T., Bauer, R., Lehmann, C., Eckardt, F., and Hoch, M. (2001). Control of endoreduplication domains in the Drosophila gut by the knirps and knirpsrelated genes. Mechs. Dev. 100, 15–23. Gabay, L., Seger, R., and Shilo, B.-Z. (1997). In situ activation pattern of Drosophila EGF receptor pathway during development. Science 277, 1103–1106. Gabay, L., Seger, R., and Shilo, B.-Z. (1997). MAP kinase in situ activation atlas during Drosophila embryogenesis. Development 124, 3535–3541. Gajewski, K., Choi, C.Y., Kim, Y., and Schulz, R.A. (2000). Genetically distinct cardial cells within the Drosophila heart. Genesis 28, 36–43. Galant, R., Skeath, J.B., Paddock, S., Lewis, D.L., and Carroll, S.B. (1998). Expression pattern of a butterﬂy achaete-scute homolog reveals the homology of butterﬂy wing scales and insect sensory bristles. Curr. Biol. 8, 807–813. Galindo, M.I. and Couso, J.P. (2000). Intercalation of cell

344

1342.

1343.

1344.

1345.

1346.

1347.

1348.

1349.

1350.

1351.

1352. 1353.

1354.

1355. 1356.

1357.

1358.

1359.

REFERENCES

fates during tarsal development in Drosophila. BioEssays 22, 777–780. Gallant, P., Li, C., Montero, L., Johnston, L.A., and Eisenman, R.N. (2001). Genetic dissection of Myc function in Drosophila. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a82. Gallant, P., Shiio, Y., Cheng, P.F., Parkhurst, S.M., and Eisenman, R.N. (1996). Myc and Max homologs in Drosophila. Science 274, 1523–1527. Gallet, A., Angelats, C., Erkner, A., Charroux, B., Fasano, L., and Kerridge, S. (1999). The C-terminal domain of Armadillo binds to hypophosphorylated Teashirt to modulate Wingless signalling in Drosophila. EMBO J. 18, 2208– 2217. Gallet, A., Erkner, A., Charroux, B., Fasano, L., and Kerridge, S. (1998). Trunk-speciﬁc modulation of Wingless signalling in Drosophila by Teashirt binding to Armadillo. Curr. Biol. 8, 893–902. Galloni, M. and Edgar, B.A. (1999). Cell-autonomous and non-autonomous growth-defective mutants of Drosophila melanogaster. Development 126, 2365–2375. Ganfornina, M.D. and S´anchez, D. (1999). Generation of evolutionary novelty by functional shift. BioEssays 21, 432–439. Garc´ıa Alonso, L. and Garc´ıa-Bellido, A. (1986). Genetic analysis of Hairy-wing mutations. Roux’s Arch. Dev. Biol. 195, 259–264. Garc´ıa Alonso, L.A. and Garc´ıa-Bellido, A. (1988). Extramacrochaetae, a trans-acting gene of the achaete-scute complex of Drosophila involved in cell communication. Roux’s Arch. Dev. Biol. 197, 328–338. Garc´ıa-Alonso, L., Fetter, R.D., and Goodman, C.S. (1996). Genetic analysis of Laminin A in Drosophila: extracellular matrix containing laminin A is required for ocellar axon pathﬁnding. Development 122, 2611–2621. Garc´ıa-Alonso, L., VanBerkum, M.F.A., Grenningloh, G., Schuster, C., and Goodman, C.S. (1995). Fasciclin II controls proneural gene expression in Drosophila. Proc. Natl. Acad. Sci. USA 92, 10501–10505. ˜ Garc´ıa-Anoveros, J. and Corey, D.P. (1997). The molecules of mechanosensation. Annu. Rev. Neurosci. 20, 567–594. Garcia-Bellido, A. (1966). Changes in selective afﬁnity following transdetermination in imaginal disc cells of Drosophila melanogaster. Exp. Cell Res. 44, 382–392. Garcia-Bellido, A. (1981). From the gene to the pattern: chaeta differentiation. In “Cellular Controls in Differentiation,” (C.W. Lloyd and D.A. Rees, eds.). Acad. Pr.: New York, pp. 281–304. Garcia-Bellido, A. (1998). The engrailed story. Genetics 148, 539–544. Garc´ıa-Bellido, A. (1966). Pattern reconstruction by dissociated imaginal disk cells of Drosophila melanogaster. Dev. Biol. 14, 278–306. Garc´ıa-Bellido, A. (1972). Pattern formation in imaginal disks. In “The Biology of Imaginal Disks,” Results and Problems in Cell Differentiation, Vol. 5, (H. Ursprung and R. N¨othiger, eds.). Springer-Verlag: Berlin, pp. 59–91. Garc´ıa-Bellido, A. (1975). Genetic control of wing disc development in Drosophila. In “Cell Patterning,” Ciba Found. Symp., Vol. 29 (R. Porter and J. Rivers, eds.). Elsevier: Amsterdam, pp. 161–182. Garc´ıa-Bellido, A. (1977). Inductive mechanisms in the process of wing vein formation in Drosophila. W. Roux’s Arch. 182, 93–106.

1360. Garc´ıa-Bellido, A. (1979). Genetic analysis of the achaetescute system of Drosophila melanogaster. Genetics 91, 491–520. 1361. Garc´ıa-Bellido, A. (1983). Comparative anatomy of cuticular patterns in the genus Drosophila. In “Development and Evolution,” Symp. Brit. Soc. Dev. Biol., Vol. 6 (B.C. Goodwin, N. Holder, and C.C. Wylie, eds.). Cambridge Univ. Pr.: Cambridge, pp. 227–255. 1362. Garc´ıa-Bellido, A. (1985). Cell lineages and genes. Phil. Trans. Roy. Soc. Lond. B 312, 101–128. 1363. Garc´ıa-Bellido, A. (1986). Genetic analysis of morphogenesis. In “Genetics, Development, and Evolution,” (J.P. Gustafson, G.L. Stebbins, and F.J. Ayala, eds.). Plenum Pr.: New York, pp. 187–209. 1364. Garc´ıa-Bellido, A. (1993). Coming of age. Trends Genet. 9, 102–103. 1365. Garc´ıa-Bellido, A. and Capdevila, M.P. (1978). The initiation and maintenance of gene activity in a developmental pathway of Drosophila. In “The Clonal Basis of Development,” Symp. Soc. Dev. Biol., Vol. 36 (S. Subtelny and I.M. Sussex, eds.). Acad. Pr.: New York, pp. 3–21. 1366. Garc´ıa-Bellido, A., Cort´es, F., and Mil´an, M. (1994). Cell interactions in the control of size in Drosophila wings. Proc. Natl. Acad. Sci. USA 91, 10222–10226. 1367. Garcia-Bellido, A. and Dapena, J. (1974). Induction, detection and characterization of cell differentiation mutants in Drosophila. Molec. Gen. Genet. 128, 117–130. 1368. Garcia-Bellido, A. and de Celis, J.F. (1992). Developmental genetics of the venation pattern of Drosophila. Annu. Rev. Genet. 26, 277–304. 1369. Garc´ıa-Bellido, A., Lawrence, P.A., and Morata, G. (1979). Compartments in animal development. Sci. Am. 241 #1, 102–110. 1370. Garcia-Bellido, A. and Merriam, J.R. (1969). Cell lineage of the imaginal discs in Drosophila gynandromorphs. J. Exp. Zool. 170, 61–76. 1371. Garcia-Bellido, A. and Merriam, J.R. (1971). Clonal parameters of tergite development in Drosophila. Dev. Biol. 26, 264–276. 1372. Garcia-Bellido, A. and Merriam, J.R. (1971). Genetic analysis of cell heredity in imaginal discs of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 68, 2222–2226. 1373. Garcia-Bellido, A. and Merriam, J.R. (1971). Parameters of the wing imaginal disc development of Drosophila melanogaster. Dev. Biol. 24, 61–87. 1374. Garcia-Bellido, A. and N¨othiger, R. (1976). Maintenance of determination by cells of imaginal discs of Drosophila after dissociation and culture in vivo. W. Roux’s Arch. 180, 189–206. 1375. Garcia-Bellido, A. and Ripoll, P. (1978). Cell lineage and differentiation in Drosophila. In “Genetic Mosaics and Cell Differentiation,” Results and Problems in Cell Differentiation, Vol. 9, (W.J. Gehring, ed.). Springer-Verlag: Berlin, pp. 119–156. 1376. Garcia-Bellido, A., Ripoll, P., and Morata, G. (1973). Developmental compartmentalisation of the wing disk of Drosophila. Nature New Biol. 245, 251–253. 1377. Garcia-Bellido, A., Ripoll, P., and Morata, G. (1976). Developmental compartmentalization in the dorsal mesothoracic disc of Drosophila. Dev. Biol. 48, 132–147. 1378. Garcia-Bellido, A. and Santamaria, P. (1972). Developmental analysis of the wing disc in the mutant engrailed of Drosophila melanogaster. Genetics 72, 87–104. 1379. Garc´ıa-Bellido, A. and Santamaria, P. (1978). Developmen-

REFERENCES

1380.

1381.

1382. 1383. 1384. 1385.

1386.

1387.

1388.

1389.

1390.

1391.

1392. 1393. 1394.

1395. 1396.

1397.

1398. 1399.

1400.

tal analysis of the achaete-scute system of Drosophila melanogaster. Genetics 88, 469–486. Garc´ıa-Garc´ıa, M.J., Ramain, P., Simpson, P., and Modolell, J. (1999). Different contributions of pannier and wingless to the patterning of the dorsal mesothorax of Drosophila. Development 126, 3523–3532. Gardner, M. (1971). On cellular automata, selfreproduction, the Garden of Eden and the game “life”. Sci. Am. 224 #2, 112–117. Gardner, M. (1983). “Wheels, Life and Other Mathematical Amusements.” W. H. Freeman, New York. Gardner, T.S. and Collins, J.J. (2000). Neutralizing noise in gene networks. Nature 405, 520–521. Garen, A. (1992). Looking for the homunculus in Drosophila. Genetics 131, 5–7. Garner, C.C., Nash, J., and Huganir, R.L. (2000). PDZ domains in synapse assembly and signalling. Trends Cell Biol. 10, 274–280. Garoia, F., Guerra, D., Pezzoli, M.C., L´opez-Varea, A., Cavicchi, S., and Garc´ıa-Bellido, A. (2000). Cell behaviour of Drosophila fat cadherin mutations in wing development. Mechs. Dev. 94, 95–109. Garrell, J. and Campuzano, S. (1991). The helix-loop-helix domain: a common motif for bristles, muscles, and sex. BioEssays 13, 493–498. Garrell, J. and Modolell, J. (1990). The Drosophila extramacrochaetae locus, an antagonist of proneural genes that, like these genes, encodes a helix-loop-helix protein. Cell 61, 39–48. Garrington, T.P. and Johnson, G.L. (1999). Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr. Opin. Cell Biol. 11, 211–218. Garrity, P.A., Lee, C.-H., Salecker, I., Robertson, H.C., Desai, C.J., Zinn, K., and Zipursky, S.L. (1999). Retinal axon target selection in Drosophila is regulated by a receptor protein tyrosine phosphatase. Neuron 22, 707–717. Garrity, P.A., Rao, Y., Salecker, I., McGlade, J., Pawson, T., and Zipursky, S.L. (1996). Drosophila photoreceptor axon guidance and targeting requires the Dreadlocks SH2/SH3 adapter protein. Cell 85, 639–650. Garrity, P.A. and Zipursky, S.L. (1995). Neuronal target recognition. Cell 83, 177–185. Gasser, S.M. (2001). Positions of potential: nuclear organization and gene expression. Cell 104, 639–642. Gateff, E. (1978). Malignant and benign neoplasms of Drosophila melanogaster. In “The Genetics and Biology of Drosophila,” Vol. 2b (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 181–275. Gateff, E. (1978). Malignant neoplasms of genetic origin in Drosophila melanogaster. Science 200, 1448–1459. Gateff, E. and Schneiderman, H.A. (1974). Developmental capacities of benign and malignant neoplasms of Drosophila. W. Roux’s Arch. 176, 23–65. Gates, J. and Thummel, C.S. (2000). An enhancer trap screen for ecdysone-inducible genes required for Drosophila adult leg morphogenesis. Genetics 156, 1765– 1776. Gaul, U. and J¨ackle, H. (1990). Role of gap genes in early Drosophila development. Adv. Genet. 27, 239–275. Gaul, U., Mardon, G., and Rubin, G.M. (1992). A putative Ras GTPase activating protein acts as a negative regulator of signaling by the Sevenless receptor tyrosine kinase. Cell 68, 1007–1019. Gaunt, S.J. and Singh, P.B. (1990). Homeogene expression

345

1401.

1402.

1403.

1404.

1405.

1406.

1407. 1408.

1409. 1410.

1411.

1412.

1413. 1414. 1415. 1416.

1417. 1418. 1419.

1420.

patterns and chromosomal imprinting. Trends Genet. 6, 208–212. Gause, M., Hovhannisyan, H., Kan, T., Kuhﬁttig, S., Mogila, V., and Georgiev, P. (1998). hobo induced rearrangements in the yellow locus inﬂuence the insulation effect of the gypsy su(Hw)-binding region in Drosophila melanogaster. Genetics 149, 1393–1405. Gautier, P., Ledent, V., Massaer, M., Dambly-Chaudi`ere, C., and Ghysen, A. (1997). tap, a Drosophila bHLH gene expressed in chemosensory organs. Gene 191, 15–21. Gayko, U., Cleghon, V., Copeland, T., Morrison, D.K., and Perrimon, N. (1999). Synergistic activities of multiple phosphotyrosine residues mediate full signaling from the Drosophila Torso receptor tyrosine kinase. Proc. Natl. Acad. Sci. USA 96, 523–528. ¨ ¨ Gehring, W. (1966). Ubertragung und Anderung der Determinationsqualit¨aten in Antennensheiben-Kulturen von Drosophila melanogaster. J. Embryol. Exp. Morph. 15, 77– 111. Gehring, W. (1967). Clonal analysis of determination dynamics in cultures of imaginal disks in Drosophila melanogaster. Dev. Biol. 16, 438–456. Gehring, W. (1972). The stability of the determined state in cultures of imaginal disks in Drosophila. In “The Biology of Imaginal Disks,” Results and Problems in Cell Differentiation, Vol. 5, (H. Ursprung and R. N¨othiger, eds.). SpringerVerlag: Berlin, pp. 35–58. Gehring, W. (2000). Reply to Meyer-Rochow. Trends Genet. 16, 245. Gehring, W. and Seippel, S. (1967). Die Imaginalzellen ¨ des Clypeo-Labrums und die Bildung des Russels von Drosophila melanogaster. Rev. Suisse Zool. 74, 589–596. Gehring, W.J. (1976). Developmental genetics of Drosophila. Annu. Rev. Genet. 10, 209–252. Gehring, W.J., ed. (1978). “Genetic Mosaics and Cell Differentiation.” Results and Problems in Cell Differentiation, Vol. 9, Springer-Verlag, Berlin. Gehring, W.J. (1978). Imaginal discs: determination. In “The Genetics and Biology of Drosophila,” Vol. 2c (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 511–554. Gehring, W.J. (1985). Homeotic genes, the homeo box, and the genetic control of development. Cold Spr. Harb. Symp. Quant. Biol. 50, 243–251. Gehring, W.J. (1985). The molecular basis of development. Sci. Am. 253 #4, 153–162. Gehring, W.J. (1987). Homeo boxes in the study of development. Science 236, 1245–1252. Gehring, W.J. (1996). The master control gene for morphogenesis and evolution of the eye. Genes to Cells 1, 11–15. Gehring, W.J. (1998). “Master Control Genes in Development and Evolution: The Homeobox Story.” Yale Univ. Pr., New Haven. [See also 2001 BioEssays 23, 763–766.] ¨ Gehring, W.J., Affolter, M., and Burglin, T. (1994). Homeodomain proteins. Annu. Rev. Biochem. 63, 487–526. Gehring, W.J. and Hiromi, Y. (1986). Homeotic genes and the homeobox. Annu. Rev. Genet. 20, 147–173. Gehring, W.J. and Ikeo, K. (1999). Pax6: mastering eye morphogenesis and eye evolution. Trends Genet. 15, 371– 377. ¨ Gehring, W.J., Muller, M., Affolter, M., Percival-Smith, A., ¨ Billeter, M., Qian, Y.Q., Otting, G., and Wuthrich, K. (1990). The structure of the homeodomain and its functional implications. Trends Genet. 6, 323–329.

346

1421. Gehring, W.J. and N¨othiger, R. (1973). The imaginal discs of Drosophila. In “Developmental Systems: Insects,” Vol. 2 (S.J. Counce and C.H. Waddington, eds.). Acad. Pr.: New York, pp. 211–290. 1422. Gehring, W.J., Qian, Y.Q., Billeter, M., FurukuboTokunaga, K., Schier, A.F., Resendez-Perez, D., Affolter, ¨ M., Otting, G., and Wuthrich, K. (1994). HomeodomainDNA recognition. Cell 78, 211–223. 1423. Gehring, W.J. and Schubiger, G. (1975). Expression of homeotic mutations in duplicated and regenerated antennae of Drosophila melanogaster. J. Embryol. Exp. Morph. 33, 459–469. 1424. Geigy, R. (1931). Erzeugung rein imaginaler Defekte durch ultraviolette Eibestrahlung bei Drosophila melanogaster. Roux’ Arch. Entw. Mech. 125, 406–447. 1425. Gekakis, N., Saez, L., Delahaye-Brown, A.-M., Myers, M.P., Sehgal, A., Young, M.W., and Weitz, C.J. (1995). Isolation of timeless by PER protein interaction: defective interaction between timeless protein and long-period mutant PER. Science 270, 811–815. 1426. Gelbart, W.M. (1982). Synapsis-dependent allelic complementation at the decapentaplegic gene complex in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 79, 2636–2640. 1427. Gelbart, W.M. (1989). The decapentaplegic gene: a TGF-β homologue controlling pattern formation in Drosophila. Development 107 (Suppl.), 65–74. 1428. Gelbart, W.M., Irish, V.F., St. Johnston, R.D., Hoffmann, F.M., Blackman, R.K., Segal, D., Posakony, L.M., and Grimaila, R. (1985). The Decapentaplegic Gene Complex in Drosophila melanogaster. Cold Spr. Harb. Symp. Quant. Biol. 50, 119–125. 1429. Gellon, G. and McGinnis, W. (1998). Shaping animal body plans in development and evolution by modulation of Hox expression patterns. BioEssays 20, 116–125. 1430. Geng, W., He, B., Wang, M., and Adler, P.N. (2000). The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase. Genetics 156, 1817–1828. 1431. Genova, J.L., Jong, S., Camp, J.T., and Fehon, R.G. (2000). Functional analysis of Cdc42 in actin ﬁlament assembly, epithelial morphogenesis, and cell signaling during Drosophila development. Dev. Biol. 221, 181–194. 1432. Georgakopoulos, A., Marambaud, P., Efthimiopoulos, S., ¨ Shioi, J., Cui, W., Li, H.-C., Schutte, M., Gordon, R., Holstein, G.R., Martinelli, G., Mehta, P., Friedrich, V.L., Jr., and Robakis, N.K. (1999). Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts. Molec. Cell 4, 893–902. 1433. George, F.H. (1965). “Cybernetics and Biology.” Oliver and Boyd, London. 1434. George, H. and Terracol, R. (1997). The vrille gene of Drosophila is a maternal enhancer of decapentaplegic and encodes a new member of the bZIP family of transcription factors. Genetics 146, 1345–1363. 1435. Georgiev, P., Tikhomirova, T., Yelagin, V., Belenkaya, T., Gracheva, E., Parshikov, A., Evgen’ev, M.B., Samarina, O.P., and Corces, V.G. (1997). Insertions of hybrid P elements in the yellow gene of Drosophila cause a large variety of mutant phenotypes. Genetics 146, 583–594. 1436. Gerasimova, T.I. and Corces, V.G. (1998). Polycomb and trithorax group proteins mediate the function of a chromatin insulator. Cell 92, 511–521. 1437. Gerhardt, M., Schuster, H., and Tyson, J.J. (1990). A cellular

REFERENCES

1438. 1439.

1440. 1441.

1442.

1443.

1444. 1445.

1446.

1447.

1448.

1449.

1450.

1451.

1452.

1453. 1454.

1455.

1456.

automaton model of excitable media including curvature and dispersion. Science 247, 1563–1566. Gerhart, J. (1989). The primacy of cell interactions in development. Trends Genet. 5, 233–236. Gerhart, J. (2000). Inversion of the chordate body axis: are there alternatives? Proc. Natl. Acad. Sci. USA 97, 4445– 4448. Gerhart, J. and Kirschner, M. (1997). “Cells, Embryos, and Evolution.” Blackwell Science, Malden, Mass. Geyer, P.K. (1997). The role of insulator elements in deﬁning domains of gene expression. Curr. Opin. Gen. Dev. 7, 242–248. Geyer, P.K. and Corces, V.G. (1987). Separate regulatory elements are responsible for the complex pattern of tissue-speciﬁc and developmental transcription of the yellow locus in Drosophila melanogaster. Genes Dev. 1, 996–1004. Geyer, P.K. and Corces, V.G. (1992). DNA position-speciﬁc repression of transcription by a Drosophila zinc ﬁnger protein. Genes Dev. 6, 1865–1873. Ghazi, A. and VijayRaghavan, K. (2000). Control by combinatorial codes. Nature 408, 419–420. Ghiglione, C., Carraway, K.L., III, Amundadottir, L.T., Boswell, R.E., Perrimon, N., and Duffy, J.B. (1999). The transmembrane molecule Kekkon 1 acts in a feedback loop to negatively regulate the activity of the Drosophila EGF receptor during oogenesis. Cell 96, 847–856. Ghiglione, C., Perrimon, N., and Perkins, L.A. (1999). Quantitative variations in the level of MAPK activity control patterning of the embryonic termini in Drosophila. Dev. Biol. 205, 181–193. Gho, M., Bella¨ıche, Y., and Schweisguth, F. (1999). Revisiting the Drosophila microchaete lineage: a novel intrinsically asymmetric cell division generates a glial cell. Development 126, 3573–3584. Gho, M., Lecourtois, M., G´eraud, G., Posakony, J.W., and Schweisguth, F. (1996). Subcellular localization of Suppressor of Hairless in Drosophila sense organ cells during Notch signaling. Development 122, 1673–1682. Gho, M. and Schweisguth, F. (1998). Frizzled signalling controls orientation of asymmetric sense organ precursor cell divisions in Drosophila. Nature 393, 178–181. Ghosh, R.N., Chen, Y.-T., DeBiasio, R., DeBiasio, R.L., Conway, B.R., Minor, L.K., and Demarest, K.T. (2000). Cellbased, high-content screen for receptor internalization, recycling and intracellular trafﬁcking. BioTechniques 29, 170–175. Ghysen, A. (1980). The projection of sensory neurons in the central nervous system of Drosophila: choice of the appropriate pathway. Dev. Biol. 78, 521–541. Ghysen, A., ed. (1998). “Developmental Genetics of Drosophila.” Int. J. Dev. Biol. 42 #3, Univ. Basque Country Pr., Bilbao, Spain. Ghysen, A. and Dambly-Chaudi`ere, C. (1988). From DNA to form: the achaete-scute complex. Genes Dev. 2, 495–501. Ghysen, A. and Dambly-Chaudi`ere, C. (1989). Genesis of the Drosophila peripheral nervous system. Trends Genet. 5, 251–255. Ghysen, A. and Dambly-Chaudi`ere, C. (1992). Development of the peripheral nervous system in Drosophila. In “Determinants of Neuronal Identity,” (M. Shankland and E.R. Macagno, eds.). Acad. Pr.: New York, pp. 225–292. Ghysen, A. and Dambly-Chaudi`ere, C. (1993). The speciﬁcation of sensory neuron identity in Drosophila. BioEssays 15, 293–298.

REFERENCES

1457. Ghysen, A. and Dambly-Chaudi`ere, C. (2000). A genetic programme for neuronal connectivity. Trends Genet. 16, 221–226. 1458. Ghysen, A., Dambly-Chaudi`ere, C., Jan, L.Y., and Jan, Y.-N. (1993). Cell interactions and gene interactions in peripheral neurogenesis. Genes Dev. 7, 723–733. 1459. Ghysen, A., Dambly-Chaudi`ere, C., Jan, L.Y., and Jan, Y.N. (1982). Segmental differences in the protein content of Drosophila imaginal discs. EMBO J. 1, 1373–1379. 1460. Ghysen, A. and Janson, R. (1980). Formation of central patterns by receptor cell axons in Drosophila. In “Development and Neurobiology of Drosophila,” (O. Siddiqi, P. Babu, L.M. Hall, and J.C. Hall, eds.). Plenum: New York, pp. 247–265. 1461. Ghysen, A., Janson, R., and Santamaria, P. (1983). Segmental determination of sensory neurons in Drosophila. Dev. Biol. 99, 7–26. 1462. Ghysen, A. and Richelle, J. (1979). Determination of sensory bristles and pattern formation in Drosophila. II. The achaete-scute locus. Dev. Biol. 70, 438–452. 1463. Giangrande, A. (1994). Glia in the ﬂy wing are clonally related to epithelial cells and use the nerve as a pathway for migration. Development 120, 523–534. 1464. Giangrande, A. (1995). Proneural genes inﬂuence gliogenesis in Drosophila. Development 121, 429–438. 1465. Giangrande, A., Murray, M.A., and Palka, J. (1993). Development and organization of glial cells in the peripheral nervous system of Drosophila melanogaster. Development 117, 895–904. 1466. Gibson, G. (1999). Redesigning the fruitﬂy. Curr. Biol. 9, R86–R89. 1467. Gibson, G. (2000). Evolution: Hox genes and the cellared wine principle. Curr. Biol. 10, R452–R455. 1468. Gibson, G. and Gehring, W.J. (1988). Head and thoracic transformations caused by ectopic expression of Antennapedia during Drosophila development. Development 102, 657–675. 1469. Gibson, G. and Hogness, D.S. (1996). Effect of polymorphism in the Drosophila regulatory gene Ultrabithorax on homeotic stability. Science 271, 200–203. 1470. Gibson, G. and Wagner, G. (2000). Canalization in evolutionary genetics: a stablilizing theory? BioEssays 22, 372– 380. 1471. Gibson, J.B., Parsons, P.A., and Spickett, S.G. (1961). Correlations between chaeta number and ﬂy size in Drosophila melanogaster. Heredity 16, 349–354. 1472. Gibson, M.C. and Schubiger, G. (1999). Hedgehog is required for activation of engrailed during regeneration of fragmented Drosophila imaginal discs. Development 126, 1591–1599. 1473. Gibson, M.C. and Schubiger, G. (2000). Peripodial cells regulate proliferation and patterning of Drosophila imaginal discs. Cell 103, 343–350. [See also 2001 BioEssays 23, 691–697.] 1474. Gibson, T.J., Hyv¨onen, M., Musacchio, A., Saraste, M., and Birney, E. (1994). PH domain: the ﬁrst anniversary. Trends Biochem. Sci. 19, 349–353. 1475. Giebel, B. and Campos-Ortega, J.A. (1997). Functional dissection of the Drosophila Enhancer of split protein, a suppressor of neurogenesis. Proc. Natl. Acad. Sci. USA 94, 6250–6254. 1476. Giepmans, B.N.G. and Moolenaar, W.H. (1998). The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein. Curr. Biol. 8, 931–934.

347

1477. Gierer, A. (1974). Molecular models and combinatorial principles in cell differentiation and morphogenesis. Cold Spr. Harb. Symp. Quant. Biol. 38, 951–961. 1478. Gierer, A. and Meinhardt, H. (1972). A theory of biological pattern formation. Kybernetik 12, 30–39. 1479. Gierer, A. and Meinhardt, H. (1974). Biological pattern formation involving lateral inhibition. In “Lectures on Mathematics in the Life Sciences,” Vol. 7 Am. Math. Soc.: Providence, Rhode Island, pp. 163–183. 1480. Giese, K., Cox, J., and Grosschedl, R. (1992). The HMG domain of Lymphoid Enhancer Factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures. Cell 69, 185–195. 1481. Giese, K., Pagel, J., and Grosschedl, R. (1997). Functional analysis of DNA bending and unwinding by the high mobility group domain of LEF-1. Proc. Natl. Acad. Sci. USA 94, 12845–12850. 1482. Gieseler, K., Graba, Y., Mariol, M.-C., Wilder, E.L., Martinez-Arias, A., Lamaire, P., and Pradel, J. (1999). Antagonist activity of DWnt-4 and wingless in the Drosophila embryonic ventral ectoderm and in heterologous Xenopus assays. Mechs. Dev. 85, 123–131. [See also 2001 Dev. Biol. 232, 339–350.] 1483. Giesen, K., Hummel, T., Stollewerk, A., Harrison, S., Travers, A., and Kl¨ambt, C. (1997). Glial development in the Drosophila CNS requires concomitant activation of glial and repression of neuronal differentiation genes. Development 124, 2307–2311. 1484. Gigliani, F., Longo, F., Gaddini, L., and Battaglia, P.A. (1996). Interactions among the bHLH domains of the proteins encoded by the Enhancer of split and achaete-scute gene complexes of Drosophila. Mol. Gen. Genet. 251, 628– 634. 1485. Gilbert, L.I. (1994). Howard A. Schneiderman (February 9, 1927–December 5, 1990). Biogr. Memoirs Natl. Acad. Sci. U. S. A. 63, 481–502. 1486. Gilbert, S.F. (1991). Induction and the origins of developmental genetics. In “A Conceptual History of Modern Embryology,” Developmental Biology: A Comprehensive Synthesis, Vol. 7 (S.F. Gilbert, ed.). Plenum: New York, pp. 181–206. 1487. Gilbert, S.F. (2000). “Developmental Biology”. 6th ed. Sinauer, Sunderland, Mass. 1488. Gilbert, S.F. (2000). Diachronic biology meets evo-devo: C. H. Waddington’s approach to evolutionary developmental biology. Amer. Zool. 40, 729–737. 1489. Gilbert, S.F. and Sarkar, S. (2000). Embracing complexity: organicism for the 21st century. Dev. Dynamics 219, 1–9. 1490. Gildea, J.J., Lopez, R., and Shearn, A. (2000). A screen for new trithorax group genes identiﬁed little imaginal discs, the Drosophila melanogaster homologue of human retinoblastoma binding protein 2. Genetics 156, 645–663. 1491. Gindhart, J.G., Jr. and Kaufman, T.C. (1995). Identiﬁcation of Polycomb and trithorax group responsive elements in the regulatory region of the Drosophila homeotic gene Sex combs reduced. Genetics 139, 797–814. 1492. Ginger, R.S., Drury, L., Baader, C., Zhukovskaya, N.V., and Williams, J.G. (1998). A novel Dictyostelium cell surface protein important for both cell adhesion and cell sorting. Development 125, 3343–3352. 1493. Giniger, E. (1998). A role for Abl in Notch signaling. Neuron 20, 667–681. 1494. Giraldez, A.J. and Cohen, S. (2001). Notum antagonizes wingless signaling during development. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a206.

348

1495. Girton, J.R. (1981). Pattern triplications produced by a celllethal mutation in Drosophila. Dev. Biol. 84, 164–172. 1496. Girton, J.R. (1982). Genetically induced abnormalities in Drosophila: two or three patterns? Amer. Zool. 22, 65–77. 1497. Girton, J.R. (1983). Morphological and somatic clonal analyses of pattern triplications. Dev. Biol. 99, 202–209. 1498. Girton, J.R. and Berns, M.W. (1982). Pattern abnormalities induced in Drosophila imaginal discs by an ultraviolet laser microbeam. Dev. Biol. 91, 73–77. 1499. Girton, J.R. and Bryant, P.J. (1980). The use of cell lethal mutations in the study of Drosophila development. Dev. Biol. 77, 233–243. 1500. Girton, J.R. and Jeon, S.H. (1994). Novel embryonic and adult homeotic phenotypes are produced by pleiohomeotic mutations in Drosophila. Dev. Biol. 161, 393– 407. 1501. Girton, J.R. and Kumor, A.L. (1985). The role of cell death in the induction of pattern abnormalities in a cell-lethal mutation of Drosophila. Dev. Genet. 5, 93–102. 1502. Girton, J.R., Langner, K., and Cejka, N. (1986). Developmental genetics of a P element induced allele of suppressor-of-forked in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 195, 334–337. 1503. Girton, J.R. and Russell, M.A. (1980). A clonal analysis of pattern duplication in a temperature-sensitive cell-lethal mutant of Drosophila melanogaster. Dev. Biol. 77, 1–21. 1504. Girton, J.R. and Russell, M.A. (1981). An analysis of compartmentalization in pattern duplications induced by a cell-lethal mutation in Drosophila. Dev. Biol. 85, 55–64. 1505. Gisselbrecht, S., Skeath, J.B., Doe, C.Q., and Michelson, A.M. (1996). heartless encodes a ﬁbroblast growth factor receptor (DFR1/DFGF-R2) involved in the directional migration of early mesodermal cells in the Drosophila embryo. Genes Dev. 10, 3003–3017. 1506. Glass, C.K., Rose, D.W., and Rosenfeld, M.G. (1997). Nuclear receptor coactivators. Curr. Opin. Cell Biol. 9, 222–232. 1507. Glazer, L. and Shilo, B.-Z. (1991). The Drosophila FGF-R homolog is expressed in the embryonic tracheal system and appears to be required for directed tracheal cell extension. Genes Dev. 5, 697–705. 1508. Glicksman, M.A. and Brower, D.L. (1988). Misregulation of homeotic gene expression in Drosophila larvae resulting from mutations at the extra sex combs locus. Dev. Biol. 126, 219–227. 1509. Glicksman, M.A. and Brower, D.L. (1990). Persistent ectopic expression of Drosophila homeotic genes resulting from maternal deﬁciency of the extra sex combs gene product. Dev. Biol. 142, 422–431. 1510. Glise, B. and Noselli, S. (1997). Coupling of Jun aminoterminal kinase and Decapentaplegic signaling pathways in Drosophila morphogenesis. Genes Dev. 11, 1738–1747. ¨ 1511. Gloor, H. (1947). Ph¨anokopie-Versuche mit Ather an Drosophila. Rev. Suisse Zool. 54, 637–712. 1512. Glotzer, M., Murray, A.W., and Kirschner, M.W. (1991). Cyclin is degraded by the ubiquitin pathway. Nature 349, 132–138. 1513. Glover, J.N.M. and Harrison, S.C. (1995). Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA. Nature 373, 257–261. 1514. Go, M.J. and Artavanis-Tsakonas, S. (1998). A genetic screen for novel components of the Notch signaling pathway during Drosophila bristle development. Genetics 150, 211–220.

REFERENCES

1515. Goberdhan, D.C.I. and Wilson, C. (1998). JNK, cytoskeletal regulator and stress response kinase? A Drosophila perspective. BioEssays 20, 1009–1019. 1516. Godt, D., Couderc, J.-L., Cramton, S.E., and Laski, F.A. (1993). Pattern formation in the limbs of Drosophila: bric a` brac is expressed in both a gradient and a wave-like pattern and is required for speciﬁcation and proper segmentation of the tarsus. Development 119, 799–812. 1517. Gogarten, J.P. and Olendzenski, L. (1999). Orthologs, paralogs and genome comparisons. Curr. Opin. Gen. Dev. 9, 630–636. 1518. Goger, M., Gupta, V., Kim, W.-Y., Shigesada, K., Ito, Y., and Werner, M.H. (1999). Molecular insights into PEBP2/ CBFβ-SMMHC associated acute leukemia revealed from the structure of PEBP2/CBFβ. Nature Struct. Biol. 6, 620– 623. 1519. Gogos, J.A., Hsu, T., Bolton, J., and Kafatos, F.C. (1992). Sequence discrimination by alternatively spliced isoforms of a DNA binding zinc ﬁnger domain. Science 257, 1951– 1955. 1520. Goldschmidt, R. (1938). “Physiological Genetics.” McGraw-Hill, New York. 1521. Goldschmidt, R. (1940). “The Material Basis of Evolution.” Yale Univ. Pr., New Haven. 1522. Goldschmidt, R.B. (1949). Phenocopies. Sci. Am. 181 #10, 46–49. 1523. Goldschmidt, R.B. (1952). Homoeotic mutants and evolution. Acta Biotheor. 10, 87–104. 1524. Goldschmidt, R.B. (1955). “Theoretical Genetics.” Univ. Calif. Pr., Berkeley. 1525. Goldstein, B. (2000). When cells tell their neighbors which direction to divide. Dev. Dynamics 218, 23–29. 1526. Goldstein, R.E., Jim´enez, G., Cook, O., Gur, D., and Paroush, Z. (1999). Huckebein repressor activity in Drosophila terminal patterning is mediated by Groucho. Development 126, 3747–3755. 1527. Golembo, M., Raz, E., and Shilo, B.-Z. (1996). The Drosophila embryonic midline is the site of Spitz processing, and induces activation of the EGF receptor in the ventral ectoderm. Development 122, 3363–3370. 1528. Golembo, M., Schweitzer, R., Freeman, M., and Shilo, B.-Z. (1996). argos transcription is induced by the Drosophila EGF receptor pathway to form an inhibitory feedback loop. Development 122, 223–230. 1529. Golembo, M., Yarnitzky, T., Volk, T., and Shilo, B.-Z. (1999). Vein expression is induced by the EGF receptor pathway to provide a positive feedback loop in patterning the Drosophila embryonic ventral ectoderm. Genes Dev. 13, 158–162. 1530. Golic, K.G. (1991). Site-speciﬁc recombination between homologous chromosomes in Drosophila. Science 252, 958–961. 1531. Golic, K.G. (1993). Generating mosaics by site-speciﬁc recombination. In “Cellular Interactions in Development: A Practical Approach,” (D.A. Hartley, ed.). Oxford Univ. Pr.: New York, pp. 1–31. 1532. Golic, K.G. and Lindquist, S. (1989). The FLP recombinase of yeast catalyzes site-speciﬁc recombination in the Drosophila genome. Cell 59, 499–509. 1533. Golling, G., Li, L.-H., Pepling, M., Stebbins, M., and Gergen, J.P. (1996). Drosophila homologs of the protooncogene product PEBP2/CBFβ regulate the DNAbinding properties of Runt. Mol. Cell. Biol. 16, 932–942. 1534. Golovnin, A., Gause, M., Georgieva, S., Gracheva, E., and

REFERENCES

1535.

1536.

1537.

1538.

1539.

1540.

1541.

1542.

1543.

1544.

1545.

1546.

1547.

1548.

1549.

1550.

Georgiev, P. (1999). The su(Hw) insulator can disrupt enhancer-promoter interactions when located more than 20 kilobases away from the Drosophila achaete-scute complex. Mol. Cell. Biol. 19, 3443–3456. Gomes, R., Karess, R.E., Ohkura, H., Glover, D.M., and Sunkel, C.E. (1993). Abnormal anaphase resolution (aar): a locus required for progression through mitosis in Drosophila. J. Cell Sci. 104, 583–593. G´omez-Skarmeta, J.-L., Diez del Corral, R., de la CalleMustienes, E., Ferr´es-Marc´o, D., and Modolell, J. (1996). araucan and caupolican, two members of the novel Iroquois Complex, encode homeoproteins that control proneural and vein-forming genes. Cell 85, 95–105. [See also 2001 Development 128, 2847–2855.] G´omez-Skarmeta, J.L. and Modolell, J. (1996). araucan and caupolican provide a link between compartment subdivisions and patterning of sensory organs and veins in the Drosophila wing. Genes Dev. 10, 2935–2945. G´omez-Skarmeta, J.L., Rodr´ıguez, I., Mart´ınez, C., Cul´ı, J., Ferr´es-Marc´o, D., Beamonte, D., and Modolell, J. (1995). Cis-regulation of achaete and scute: shared enhancer-like elements drive their coexpression in proneural clusters of the imaginal discs. Genes Dev. 9, 1869–1882. Gong, Q., Rangarajan, R., Seeger, M., and Gaul, U. (1999). The Netrin receptor Frazzled is required in the target for establishment of retinal projections in the Drosophila visual system. Development 126, 1451–1456. Gonz´alez, F., Romani, S., Cubas, P., Modolell, J., and Campuzano, S. (1989). Molecular analysis of the asense gene, a member of the achaete-scute complex of Drosophila melanogaster, and its novel role in optic lobe development. EMBO J. 8, 3553–3562. Gonz´alez, F., Swales, L., Bejsovec, A., Skaer, H., and Martinez Arias, A. (1991). Secretion and movement of wingless protein in the epidermis of the Drosophila embryo. Mechs. Dev. 35, 43–54. Gonz´alez-Crespo, S., Abu-Shaar, M., Torres, M., Mart´ınezA, C., Mann, R.S., and Morata, G. (1998). Antagonism between extradenticle function and Hedgehog signalling in the developing limb. Nature 394, 196–200. Gonz´alez-Crespo, S. and Morata, G. (1995). Control of Drosophila adult pattern by extradenticle. Development 121, 2117–2125. Gonz´alez-Crespo, S. and Morata, G. (1996). Genetic evidence for the subdivision of the arthropod limb into coxopodite and telopodite. Development 122, 3921–3928. Gonz´alez-Gait´an, M., Capdevila, M.P., and Garc´ıa-Bellido, A. (1994). Cell proliferation patterns in the wing imaginal disc of Drosophila. Mechs. Dev. 40, 183–200. Gonz´alez-Gait´an, M. and J¨ackle, H. (1999). The range of spalt-activating Dpp signalling is reduced in endocytosisdefective Drosophila wing discs. Mechs. Dev. 87, 143–151. Gonz´alez-Gait´an, M.A., Micol, J.-L., and Garc´ıa-Bellido, A. (1990). Developmental genetic analysis of Contrabithorax mutations in Drosophila melanogaster. Genetics 126, 139– 155. Gonz´alez-Hoyuela, M., Barbas, J.A., and Rodr´ıguez-T´ebar, A. (2001). The autoregulation of retinal ganglion cell number. Development 128, 117–124. Gonz´alez-Reyes, A. and Morata, G. (1990). The developmental effect of overexpressing a Ubx product in Drosophila embryos is dependent on its interactions with other homeotic products. Cell 61, 515–522. Gonz´alez-Reyes, A., Urquia, N., Gehring, W.J., Struhl, G.,

349

1551.

1552.

1553.

1554.

1555.

1556. 1557. 1558. 1559.

1560.

1561.

1562.

1563.

1564.

1565. 1566. 1567.

1568. 1569.

1570.

1571.

and Morata, G. (1990). Are cross-regulatory interactions between homoeotic genes functionally signiﬁcant? Nature 344, 78–80. Goode, S. and Perrimon, N. (1997). Brainiac and Fringe are similar pioneer proteins that impart speciﬁcity to Notch signaling during Drosophila development. Cold Spr. Harb. Symp. Quant. Biol. 62, 177–184. Goodman, C.S. and Coughlin, B.C. (2000). The evolution of evo-devo biology. Proc. Natl. Acad. Sci. USA 97, 4424– 4425. Goodman, R.H. and Smolik, S. (2000). CBP/p300 in cell growth, transformation, and development. Genes Dev. 14, 1553–1577. Goodrich, L.V. and Scott, M.P. (1998). Hedgehog and Patched in neural development and disease. Neuron 21, 1243–1257. Goodsell, D.S. and Olson, A.J. (2000). Structural symmetry and protein function. Annu. Rev. Biophys. Biomol. Struct. 29, 105–153. Goodstein, J.R. (1991). “Millikan’s School.” W. W. Norton, New York. Goodwin, B. (1994). “How the Leopard Changed Its Spots.” Charles Scribner’s Sons, New York. Goodwin, B.C. (1982). Development and evolution. J. Theor. Biol. 97, 43–55. Gopal, S., Schroeder, M., Pieper, U., Sczyrba, A., AytekinKurban, G., Bekiranov, S., Fajardo, J.E., Eswar, N., Sanchez, R., Sali, A., and Gaasterland, T. (2001). Homology-based annotation yields 1,042 new candidate genes in the Drosophila melanogaster genome. Nature Genet. 27, 337– 340. Gordon, R. (1999). “The Hierarchical Genome and Differentiation Waves: Novel Uniﬁcation of Development, Genetics and Evolution.” Vols. 1 and 2. World Scientiﬁc, Singapore. Gorﬁnkiel, N., Morata, G., and Guerrero, I. (1997). The homeobox gene Distal-less induces ventral appendage development in Drosophila. Genes Dev. 11, 2259–2271. Gorﬁnkiel, N., S´anchez, L., and Guerrero, I. (1999). Drosophila terminalia as an appendage-like structure. Mechs. Dev. 86, 113–123. Goriely, A., Dumont, N., Dambly-Chaudi`ere, C., and Ghysen, A. (1991). The determination of sense organs in Drosophila: effect of the neurogenic mutations in the embryo. Development 113, 1395–1404. Gorina, S. and Pavletich, N.P. (1996). Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2. Science 274, 1001–1005. G¨orlich, D. (1997). Nuclear protein import. Curr. Op. Cell Biol. 9, 412–419. G¨orlich, D. (1998). Transport into and out of the cell nucleus. EMBO J. 17, 2721–2727. G¨orlich, D. and Kutay, U. (1999). Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660. G¨orlich, D. and Mattaj, I.W. (1996). Nucleocytoplasmic transport. Science 271, 1513–1518. G¨orlich, D., Prehn, S., Laskey, R.A., and Hartmann, E. (1994). Isolation of a protein that is essential for the ﬁrst step of nuclear protein import. Cell 79, 767–778. Gorman, M.J. and Girton, J.R. (1992). A genetic analysis of deltex and its interaction with the Notch locus in Drosophila melanogaster. Genetics 131, 99–112. Goto, S. and Hayashi, S. (1997). Cell migration within the

350

1572.

1573.

1574.

1575.

1576.

1577.

1578.

1579.

1580.

1581. 1582. 1583. 1584. 1585. 1586.

1587.

1588.

1589.

REFERENCES

embryonic limb primordium of Drosophila as revealed by a novel ﬂuorescence method to visualize mRNA and protein. Dev. Genes Evol. 207, 194–198. Goto, S. and Hayashi, S. (1997). Speciﬁcation of the embryonic limb primordium by graded activity of Decapentaplegic. Development 124, 125–132. Goto, S. and Hayashi, S. (1999). Proximal to distal cell communication in the Drosophila leg provides a basis for an intercalary mechanism of limb patterning. Development 126, 3407–3413. Goto, S., Tanimura, T., and Hotta, Y. (1995). Enhancer-trap detection of expression patterns corresponding to the polar coordinate system in the imaginal discs of Drosophila melanogaster. Roux’s Arch. Dev. Biol. 204, 378–391. Gotoh, N., Toyoda, M., and Shibuya, M. (1997). Tyrosine phosphorylation sites at amino acids 239 and 240 of Shc are involved in Epidermal growth factor-induced mitogenic signaling that is distinct from Ras/mitogenactivated protein kinase activation. Mol. Cell. Biol. 17, 1824–1831. Gottardi, C.J., Arpin, M., Fanning, A.S., and Louvard, D. (1996). The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts. Proc. Natl. Acad. Sci. USA 93, 10779–10784. Gottlieb, F.J. (1964). Genetic control of pattern determination in Drosophila. The action of Hairy-wing. Genetics 49, 739–760. G¨otz, J., Probst, A., Mistl, C., Nitsch, R.M., and Ehler, E. (2000). Distinct role of protein phosphatase 2A subunit Ca in the regulation of E-cadherin and β-catenin during development. Mechs. Dev. 93, 83–93. Gould, A., Morrison, A., Sproat, G., White, R.A.H., and Krumlauf, R. (1997). Positive cross-regulation and enhancer sharing: two mechanisms for specifying overlapping Hox expression patterns. Genes Dev. 11, 900–913. Gould, A.P., Brookman, J.J., Strutt, D.I., and White, R.A.H. (1990). Targets of homeotic gene control in Drosophila. Nature 348, 308–312. Gould, S.J. (1977). “Ontogeny and Phylogeny.” Harvard Univ. Pr., Cambridge, Mass. Gould, S.J. (1977). The return of hopeful monsters. Nat. Hist. 86 #6, 22–30. [Cf. 2001 Genetics 159, 1383–1392.] Gould, S.J. (1980). The evolutionary biology of constraint. Proc. Amer. Acad. Arts Sci. 109 #2, 39–52. Gould, S.J. (1980). Is a new and general theory of evolution emerging? Paleobiol. 6, 119–130. Gould, S.J. (1981). What color is a zebra? Nat. Hist. 90 #8, 16–22. Goulding, S.E., White, N.M., and Jarman, A.P. (2000). cato encodes a basic helix-loop-helix transcription factor implicated in the correct differentiation of Drosophila sense organs. Dev. Biol. 221, 120–131. Goulding, S.E., zur Lage, P., and Jarman, A.P. (2000). amos, a proneural gene for Drosophila olfactory sense organs that is regulated by lozenge. Neuron 25, 69–78. Gout, I., Dhand, R., Hiles, I.D., Fry, M.J., Panayotou, G., Das, P., Truong, O., Totty, N.F., Hsuan, J., Booker, G.W., Campbell, I.D., and Waterﬁeld, M.D. (1993). The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell 75, 25–36. Gowher, H., Leismann, O., and Jeltsch, A. (2000). DNA of Drosophila melanogaster contains 5-methylcytosine. EMBO J. 19, 6918–6923.

1590. Goyal, L., McCall, K., Agapite, J., Hartwieg, E., and Steller, H. (2000). Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function. EMBO J. 19, 589–597. 1591. Graba, Y., Aragnol, D., and Pradel, J. (1997). Drosophila Hox complex downstream targets and the function of homeotic genes. BioEssays 19, 379–388. 1592. Graba, Y., Gieseler, K., Aragnol, D., Laurenti, P., Mariol, M.C., Berenger, H., Sagnier, T., and Pradel, J. (1995). DWnt-4, a novel Drosophila Wnt gene acts downstream of homeotic complex genes in the visceral mesoderm. Development 121, 209–218. 1593. Graham, T. A., Weaver, C., Mao, F., Kimelman, D., and Xu, W. (2000). Crystal structure of a β-catenin/Tcf complex. Cell 103, 885–896. 1594. Grandori, C., Cowley, S.M., James, L.P., and Eisenman, R.N. (2000). The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu. Rev. Cell Dev. Biol. 16, 653–699. 1595. Grant, D., Unadkat, S., Katzen, A., Krishnan, K.S., and Ramaswami, M. (1998). Probable mechanisms underlying interallelic complementation and temperaturesensitivity of mutations at the shibire locus of Drosophila melanogaster. Genetics 149, 1019–1030. 1596. Grau, Y. and Simpson, P. (1987). The segment polarity gene costal-2 in Drosophila. I. The organization of both primary and secondary embryonic ﬁelds may be affected. Dev. Biol. 122, 186–200. 1597. Graveley, B.R. (2001). Alternative splicing: increasing diversity in the proteomic world. Trends Genet. 17, 100–107. 1598. Graves, B. and Schubiger, G. (1981). Regional differences in the developing foreleg of Drosophila melanogaster. Dev. Biol. 85, 334–343. 1599. Graves, B.J. and Schubiger, G. (1982). Cell cycle changes during growth and differentiation of imaginal leg discs in Drosophila melanogaster. Dev. Biol. 93, 104–110. 1600. Gray, S. and Levine, M. (1996). Short-range transcriptional repressors mediate both quenching and direct repression within complex loci in Drosophila. Genes Dev. 10, 700–710. 1601. Gray, S., Szymanski, P., and Levine, M. (1994). Short-range repression permits multiple enhancers to function autonomously within a complex promoter. Genes Dev. 8, 1829–1838. 1602. Gray, Y.H.M. (2000). It takes two transposons to tango: transposable-element-mediated chromosomal rearrangements. Trends Genet. 16, 461–468. 1603. Greaves, S., Sanson, B., White, P., and Vincent, J.-P. (1999). A screen for identifying genes interacting with armadillo, the Drosophila homolog of β-catenin. Genetics 153, 1753– 1766. 1604. Greco, V., Hannus, M., and Eaton, S. (2001). Argosomes: a potential vehicle for the spread of morphogens through epithelia. Cell 106, 633–645. 1605. Green, D.R. (2000). Apoptotic pathways: paper wraps stone blunts scissors. Cell 102, 1–4. 1606. Green, H. and Thomas, J. (1978). Pattern formation by cultured human epidermal cells: development of curved ridges resembling dermatoglyphics. Science 200, 1385– 1388. 1607. Green, J.B.A. and Cooke, J. (1991). Induction, gradient models and the role of negative feedback in body pattern formation in the amphibian embryo. Sems. Dev. Biol. 2, 95–106. 1608. Green, J.B.A. and Smith, J.C. (1991). Growth factors as

REFERENCES

1609.

1610. 1611.

1612. 1613. 1614.

1615.

1616.

1617.

1618.

1619.

1620.

1621.

1622.

1623.

1624.

1625.

morphogens: do gradients and thresholds establish body plan? Trends Genet. 7, 245–250. Green, J.B.A., Smith, J.C., and Gerhart, J.C. (1994). Slow emergence of a multithreshold response to activin requires cell-contact-dependent sharpening but not prepattern. Development 120, 2271–2278. Green, P.B. (1980). Organogenesis–a biophysical view. Annu. Rev. Plant Physiol. 31, 51–82. Greenberg, R.M. and Adler, P.N. (1982). Protein synthesis and accumulation in Drosophila melanogaster imaginal discs: identiﬁcation of a protein with a nonrandom spatial distribution. Dev. Biol. 89, 273–286. Greenwald, I. (1994). Structure/function studies of lin12/Notch proteins. Curr. Op. Gen. Dev. 4, 556–562. Greenwald, I. (1998). LIN-12/Notch signaling: lessons from worms and ﬂies. Genes Dev. 12, 1751–1762. Greenwald, I. and Rubin, G.M. (1992). Making a difference: the role of cell-cell interactions in establishing separate identities for equivalent cells. Cell 68, 271–281. Greenwood, S. and Struhl, G. (1997). Different levels of Ras activity can specify distinct transcriptional and morphological consequences in early Drosophila embryos. Development 124, 4879–4886. Greenwood, S. and Struhl, G. (1999). Progression of the morphogenetic furrow in the Drosophila eye: the roles of Hedgehog, Decapentaplegic and the Raf pathway. Development 126, 5795–5808. Grenier, J.K. and Carroll, S.B. (2000). Functional evolution of the Ultrabithorax protein. Proc. Natl. Acad. Sci. USA 97, 704–709. Grenier, J.K., Garber, T.L., Warren, R., Whitington, P.M., and Carroll, S. (1997). Evolution of the entire arthropod Hox gene set predated the origin and radiation of the onychophoran/arthropod clade. Curr. Biol. 7, 547–553. Grenningloh, G., Bieber, A.J., Rehm, E.J., Snow, P.M., Traquina, Z.R., Hortsch, M., Patel, N.H., and Goodman, C.S. (1990). Molecular genetics of neuronal recognition in Drosophila: Evolution and function of immunoglobulin superfamily cell adhesion molecules. Cold Spr. Harb. Symp. Quant. Biol. 55, 327–340. Grenningloh, G., Rehm, E.J., and Goodman, C.S. (1991). Genetic analysis of growth cone guidance in Drosophila: Fasciclin II functions as a neuronal recognition molecule. Cell 67, 45–57. Grether, M.E., Abrams, J.M., Agapite, J., White, K., and Steller, H. (1995). The head involution defective gene of Drosophila melanogaster functions in programmed cell death. Genes Dev. 9, 1694–1708. Grieder, N.C., Nellen, D., Burke, R., Basler, K., and Affolter, M. (1995). schnurri is required for Drosophila Dpp signaling and encodes a zinc ﬁnger protein similar to the mammalian transcription factor PRDII-BF1. Cell 81, 791– 800. Grigliatti, T. and Suzuki, D.T. (1971). Temperature-sensitive mutations in Drosophila melanogaster, VIII. The homeotic mutant, ss a40a . Proc. Natl. Acad. Sci. USA 68, 1307–1311. Grillenzoni, N., van Helden, J., Dambly-Chaudi`ere, C., and Ghysen, A. (1998). The iroquois complex controls the somatotopy of Drosophila notum mechanosensory projections. Development 125, 3563–3569. Grimaldi, D.A. (1987). Amber fossil Drosophilidae (Diptera), with particular reference to the Hispaniolan taxa. Amer. Mus. Novitates 2880, 1–23.

351

1626. Grimm, S. and Pﬂugfelder, G.O. (1996). Control of the gene optomotor-blind in Drosophila wing development by decapentaplegic and wingless. Science 271, 1601–1604. 1627. Griswold-Prenner, I., Kamibayashi, C., Maruoka, E.M., Mumby, M.C., and Derynck, R. (1998). Physical and functional interactions between type I transforming growth factor β receptors and Bα, a WD-40 repeat subunit of phosphatase 2A. Mol. Cell. Biol. 18, 6595–6604. 1628. Gritzan, U., Hatini, V., and DiNardo, S. (1999). Mutual antagonism between signals secreted by adjacent Wingless and Engrailed cells leads to speciﬁcation of complementary regions of the Drosophila parasegment. Development 126, 4107–4115. 1629. Grosschedl, R., Giese, K., and Pagel, J. (1994). HMG domain proteins: architectural elements in the assembly of nucleoprotein structures. Trends Genet. 10, 94–100. 1630. Grossman, J., ed. (1993). “The Chicago Manual of Style.” 14th ed. Univ. Chicago Pr., Chicago. 1631. Grossniklaus, U., Cadigan, K.M., and Gehring, W.J. (1994). Three maternal coordinate systems cooperate in the patterning of the Drosophila head. Development 120, 3155– 3171. 1632. Gruss, P. and Walther, C. (1992). Pax in development. Cell 69, 719–722. [Cf. 2002 Trends Genet. 18, 41–47.] 1633. Gu, W., Wei, X., Pannuti, A., and Lucchesi, J.C. (2000). Targeting the chromatin-remodeling MSL complex of Drosophila to its sites of action on the X chromosome requires both acetyl transferase and ATPase activities. EMBO J. 19, 5202–5211. [ See also 2001 Dev. Biol. 234, 275–288.] 1634. Gu, Y., Hukriede, N.A., and Fleming, R.J. (1995). Serrate expression can functionally replace Delta activity during neuroblast segregation in the Drosophila embryo. Development 121, 855–865. 1635. Gubb, D. (1985). Domains, compartments and determinative switches in Drosophila development. BioEssays 2, 27–31. 1636. Gubb, D. (1985). Further studies on engrailed mutants in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 194, 236– 246. 1637. Gubb, D. (1986). Turing’s ﬂy. Nature 323, 675. 1638. Gubb, D. (1993). Genes controlling cellular polarity in Drosophila. Development 1993 Suppl., 269–277. 1639. Gubb, D. (1998). Cellular polarity, mitotic synchrony and axes of symmetry during growth. Where does the information come from? Int. J. Dev. Biol. 42, 369–377. 1640. Gubb, D. and Garc´ıa-Bellido, A. (1982). A genetic analysis of the determination of cuticular polarity during development in Drosophila melanogaster. J. Embryol. Exp. Morph. 68, 37–57. 1641. Gubb, D., Green, C., Huen, D., Coulson, D., Johnson, G., Tree, D., Collier, S., and Roote, J. (1999). The balance between isoforms of the Prickle LIM domain protein is critical for planar polarity in Drosophila imaginal discs. Genes Dev. 13, 2315–2327. 1642. Guichard, A., Bergeret, E., and Grifﬁn-Shea, R. (1997). Overexpression of RnRacGAP in Drosophila melanogaster deregulates cytoskeletal organisation in cellularising embryos and induces discrete imaginal phenotypes. Mechs. Dev. 61, 49–62. 1643. Guichard, A., Biehs, B., Sturtevant, M.A., Wickline, L., Chacko, J., Howard, K., and Bier, E. (1999). rhomboid and Star interact synergistically to promote EGFR/MAPK signaling during Drosophila wing vein development. Development 126, 2663–2676.

352

1644. Guichard, A., Roark, M., Ronshaugen, M., and Bier, E. (2000). brother of rhomboid, a rhomboid-related gene expressed during early Drosophila oogenesis, promotes EGF-R/MAPK signaling. Dev. Biol. 226, 255–266. 1645. Guild, G.M., Connelly, P.S., Vranich, K.A., Shaw, M.K., and Tilney, L.G. (2001). Actin ﬁlament turnover removes bundles from Drosophila bristle cells. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a75. ¨ 1646. Guillemin, K., Groppe, J., Ducker, K., Treisman, R., Hafen, E., Affolter, M., and Krasnow, M.A. (1996). The pruned gene encodes the Drosophila serum response factor and regulates cytoplasmic outgrowth during terminal branching of the tracheal system. Development 122, 1353– 1362. 1647. Guill´en, I., Mullor, J.L., Capdevila, J., S´anchez-Herrero, E., Morata, G., and Guerrero, I. (1995). The function of engrailed and the speciﬁcation of Drosophila wing pattern. Development 121, 3447–3456. 1648. Gumbiner, B.M. (1993). Proteins associated with the cytoplasmic surface of adhesion molecules. Neuron 11, 551– 564. 1649. Gumbiner, B.M. (1998). Propagation and localization of Wnt signaling. Curr. Opin. Gen. Dev. 8, 430–435. 1650. Guo, M., Bier, E., Jan, L.Y., and Jan, Y.N. (1995). tramtrack acts downstream of numb to specify distinct daughter cell fates during asymmetric cell divisions in the Drosophila PNS. Neuron 14, 913–925. 1651. Guo, M., Jan, L.Y., and Jan, Y.N. (1996). Control of daughter cell fates during asymmetric division: Interaction of Numb and Notch. Neuron 17, 27–41. 1652. Guo, Y., Livne-Bar, I., Zhou, L., and Boulianne, G.L. (1999). Drosophila presenilin is required for neuronal differentiation and affects Notch subcellular localization and signaling. J. Neurosci. 19, 8435–8442. 1653. Gupta, B.P., Flores, G.V., Banerjee, U., and Rodrigues, V. (1998). Patterning an epidermal ﬁeld: Drosophila Lozenge, a member of the AML-1/Runt family of transcription factors, speciﬁes olfactory sense organ type in a dosedependent manner. Dev. Biol. 203, 400–411. 1654. Gurdon, J.B. (1992). The generation of diversity and pattern in animal development. Cell 68, 185–199. 1655. Gurdon, J.B., Dyson, S., and St. Johnston, D. (1998). Cells’ perception of position in a concentration gradient. Cell 95, 159–162. [See also 2001 references: Curr. Biol. 11, 1578– 1585; Nature 413, 797–803.] 1656. Gurdon, J.B., Harger, P., Mitchell, A., and Lemaire, P. (1994). Activin signalling and response to a morphogen gradient. Nature 371, 487–492. 1657. Gurdon, J.B., Mitchell, A., and Mahony, D. (1995). Direct and continuous assessment by cells of their position in a morphogen gradient. Nature 376, 520–521. 1658. Gurganus, M.C., Fry, J.D., Nuzhdin, S.V., Pasyukova, E.G., Lyman, R.F., and Mackay, T.F.C. (1998). Genotypeenvironment interaction at quantitative trait loci affecting sensory bristle number in Drosophila melanogaster. Genetics 149, 1883–1898. 1659. Gustavson, E., Goldsborough, A.S., Ali, Z., and Kornberg, T.B. (1996). The Drosophila engrailed and invected genes: partners in regulation, expression and function. Genetics 142, 893–906. 1660. Gutjahr, T., Frei, E., Spicer, C., Baumgartner, S., White, R.A.H., and Noll, M. (1995). The Polycomb-group gene, extra sex combs, encodes a nuclear member of the WD-40 repeat family. EMBO J. 14, 4296–4306.

REFERENCES

1661. Gutowitz, H., ed. (1991). “Cellular Automata: Theory and Experiment.” M.I.T. Pr., Cambridge, Mass. 1662. Haas, H.J. (1968). On the epigenetic mechanisms of patterns in the insect integument. (A reappraisal of older concepts). Int. Rev. Gen. Exp. Zool. 3, 1–51. 1663. Hackel, P.O., Zwick, E., Prenzel, N., and Ullrich, A. (1999). Epidermal growth factor receptors: critical mediators of multiple receptor pathways. Curr. Opin. Cell Biol. 11, 184– 189. 1664. H¨acker, U., Lin, X., and Perrimon, N. (1997). The Drosophila sugarless gene modulates Wingless signaling and encodes an enzyme involved in polysaccharide biosynthesis. Development 124, 3565–3573. 1665. Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y., and Krasnow, M.A. (1998). sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell 92, 253–263. 1666. Hadley, N.F. (1986). The arthropod cuticle. Sci. Am. 255 #1, 104–112. 1667. Hadorn, E. (1961). “Developmental Genetics and Lethal Factors.” Methuen, London (transl. fr. 1955 German original, Thieme Verlag, Stuttgart, by U. Mittwoch). 1668. Hadorn, E. (1965). Problems of determination and transdetermination. Brookhaven Symp. Biol. 18, 148–161. 1669. Hadorn, E. (1968). Transdetermination in cells. Sci. Am. 219 #11, 110–120. 1670. Hadorn, E. (1978). Transdetermination. In “The Genetics and Biology of Drosophila,” Vol. 2c (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 555–617. 1671. Haenlin, M., Cubadda, Y., Blondeau, F., Heitzler, P., Lutz, Y., Simpson, P., and Ramain, P. (1997). Transcriptional activity of Pannier is regulated negatively by heterodimerization of the GATA DNA-binding domain with a cofactor encoded by the u-shaped gene of Drosophila. Genes Dev. 11, 3096– 3108. 1672. Haenlin, M., Kunisch, M., Kramatschek, B., and CamposOrtega, J.A. (1994). Genomic regions regulating early embryonic expression of the Drosophila neurogenic gene Delta. Mechs. Dev. 47, 99–110. 1673. Haerry, T.E., Heslip, T.R., Marsh, J.L., and O’Connor, M.B. (1997). Defects in glucuronate biosynthesis disrupt Wingless signaling in Drosophila. Development 124, 3055–3064. 1674. Haerry, T.E., Khalsa, O., O’Connor, M.B., and Wharton, K.A. (1998). Synergistic signaling by two BMP ligands through the SAX and TKV receptors controls wing growth and patterning in Drosophila. Development 125, 3977–3987. 1675. Hafen, E. (1998). Kinases and phosphatases – a marriage is consummated. Science 280, 1212–1213. 1676. Hafen, E. and Basler, K. (1990). Mechanisms of positional signalling in the developing eye of Drosophila studied by ectopic expression of sevenless and rough. In “Growth Factors in Cell and Developmental Biology,” J. Cell Sci. Suppl., Vol. 13 (M.D. Waterﬁeld, ed.). Company of Biologists, Ltd.: Cambridge, pp. 157–168. 1677. Hafen, E., Basler, K., Edstroem, J.-E., and Rubin, G.M. (1987). Sevenless, a cell-speciﬁc homeotic gene of Drosophila, encodes a putative transmembrane receptor with a tyrosine kinase domain. Science 236, 55–63. 1678. Hafen, E., Dickson, B., Raabe, T., Brunner, D., Oellers, N., and van der Straten, A. (1993). Genetic analysis of the sevenless signal transduction pathway of Drosophila. Development 1993 Suppl., 41–46. 1679. Hagman, J., Gutch, M.J., Lin, H., and Grosschedl, R. (1995). EBF contains a novel zinc coordination motif and

REFERENCES

1680.

1681.

1682.

1683.

1684.

1685.

1686.

1687.

1688.

1689. 1690.

1691. 1692. 1693.

1694.

1695.

1696.

multiple dimerization and transcriptional activation domains. EMBO J. 14, 2907–2916. Hagstrom, K., Muller, M., and Schedl, P. (1996). Fab-7 functions as a chromatin domain boundary to ensure proper segment speciﬁcation by the Drosophila bithorax complex. Genes Dev. 10, 3202–3215. Hagstrom, K., Muller, M., and Schedl, P. (1997). A Polycomb and GAGA dependent silencer adjoins the Fab-7 boundary in the Drosophila bithorax complex. Genetics 146, 1365–1380. Hagting, A., Jackman, M., Simpson, K., and Pines, J. (1999). Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal. Curr. Biol. 9, 680–689. Haines, N. and van den Heuvel, M. (2000). A directed mutagenesis screen in Drosophila melanogaster reveals new mutants that inﬂuence hedgehog signaling. Genetics 156, 1777–1785. Halder, G., Callaerts, P., Flister, S., Walldorf, U., Kloter, U., and Gehring, W.J. (1998). Eyeless initiates the expression of both sine oculis and eyes absent during Drosophila compound eye development. Development 125, 2181–2191. Halder, G., Callaerts, P., and Gehring, W.J. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267, 1788–1792. Halder, G., Polaczyk, P., Kraus, M.E., Hudson, A., Kim, J., Laughon, A., and Carroll, S. (1998). The Vestigial and Scalloped proteins act together to directly regulate wing-speciﬁc gene expression in Drosophila. Genes Dev. 12, 3900–3909. [See also 2001 references: Development 128, 3295–3305; Science 292, 1080–1081; Science 292, 1164–1167.] Halfar, K., Rommel, C., Stocker, H., and Hafen, E. (2001). Ras controls growth, survival and differentiation in the Drosophila eye by different thresholds of MAP kinase activity. Development 128, 1687–1696. Halfon, M.S., Carmena, A., Gisselbrecht, S., Sackerson, C.M., Jim´enez, F., Baylies, M.K., and Michelson, A.M. (2000). Ras pathway speciﬁcity is determined by the integration of multiple signal-activated and tissue-restricted transcription factors. Cell 103, 63–74. Hall, A. (1994). A biochemical function for Ras–at last. Science 264, 1413–1414. Hall, B.K. (1983). Epigenetic control in development and evolution. In “Development and Evolution,” (B.C. Goodwin, N. Holder, and C.G. Wylie, eds.). Cambridge Univ. Pr.: Cambridge, pp. 353–379. Hall, B.K. (2000). Evo-devo or devo-evo–does it matter? Evol. Dev. 2, 177–178. Hall, J.C. (1994). The mating of a ﬂy. Science 264, 1702– 1714. Hall, J.C. (1995). Tripping along the trail to the molecular mechanisms of biological clocks. Trends Neurosci. 18, 230– 240. Hall, J.C. (1996). Are cycling gene products as internal zeitgebers no longer the zeitgeist of chronobiology? Neuron 17, 799–802. Hall, J.C., Gelbart, W.M., and Kankel, D.R. (1976). Mosaic systems. In “The Genetics and Biology of Drosophila,” Vol. 1a (M. Ashburner and E. Novitski, eds.). Acad. Pr.: New York, N. Y., pp. 265–314. Halle, J.-P. and Meisterernst, M. (1996). Gene expression: increasing evidence for a transcriptosome. Trends Genet. 12, 161–163.

353

1697. Hama, C., Ali, Z., and Kornberg, T.B. (1990). Regionspeciﬁc recombination and expression are directed by portions of the Drosophila engrailed promoter. Genes Dev. 4, 1079–1093. 1698. Hamada, F., Tomoyasu, Y., Takatsu, Y., Nakamura, M., Nagai, S.-i., Suzuki, A., Fujita, F., Shibuya, H., Toyoshima, K., Ueno, N., and Akiyama, T. (1999). Negative regulation of Wingless signaling by D-Axin, a Drosophila homolog of Axin. Science 283, 1739–1742. 1699. Hamburger, V. (1988). “The Heritage of Experimental Embryology: Hans Spemann and the Organizer.” Oxford Univ. Pr., New York. 1700. Hamiche, A., Sandaltzopoulos, R., Gdula, D.A., and Wu, C. (1999). ATP-dependent histone octamer sliding mediated by the chromatin-remodeling complex NURF. Cell 97, 833–842. 1701. Hammerschmidt, M., Brook, A., and McMahon, A.P. (1997). The world according to hedgehog. Trends Genet. 13, 14–21. 1702. Hammond, S.M., Caudy, A.A., and Hannon, G.J. (2001). Post-transcriptional gene silencing by double-stranded RNA. Nature Rev. Gen. 2, 110–119. 1703. Hampsey, M. and Reinberg, D. (1999). RNA polymerase II as a control panel for multiple coactivator complexes. Curr. Opin. Gen. Dev. 9, 132–139. 1704. Han, J.-D., Baker, N.E., and Rubin, C.S. (1997). Molecular characterization of a novel A kinase anchor protein from Drosophila melanogaster. J. Biol. Chem. 272 #42, 26611– 26619. 1705. 1705. Han, K., Levine, M.S., and Manley, J.L. (1989). Synergistic activation and repression of transcription by Drosophila homeobox proteins. Cell 56, 573–583. 1706. Han, K. and Manley, J.L. (1993). Functional domains of the Drosophila Engrailed protein. EMBO J. 12, 2723–2733. 1707. Handford, P.A., Mayhew, M., Baron, M., Winship, P.R., Campbell, I.D., and Brownlee, G.G. (1991). Key residues involved in calcium-binding motifs in EGF-like domains. Nature 351, 164–167. 1708. Hanes, S.D. and Burz, D.S. (2001). Isolation of mutations that disrupt cooperative DNA binding by the Drosophila Bicoid protein. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a35. [See also 2001 J. Mol. Biol. 305, 219– 230.] 1709. Hanes, S.D., Riddihough, G., Ish-Horowicz, D., and Brent, R. (1994). Speciﬁc DNA recognition and intersite spacing are critical for action of the Bicoid morphogen. Mol. Cell. Biol. 14, 3364–3375. 1710. Hankinson, O. (1995). The aryl hydrocarbon receptor complex. Annu. Rev. Pharmacol. Toxicol. 35, 307–340. 1711. Hanks, S.K. and Hunter, T. (1995). The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classiﬁcation. FASEB J. 9, 576–596. 1712. Hanna-Rose, W. and Hansen, U. (1996). Active repression mechanisms of eukaryotic transcription repressors. Trends Genet. 12, 229–234. 1713. Hannah-Alava, A. (1958). Developmental genetics of the posterior legs in Drosophila melanogaster. Genetics 43, 878–905. 1714. Hannah-Alava, A. (1958). Morphology and chaeotaxy of the legs of Drosophila melanogaster. J. Morph. 103, 281– 310. 1715. Hannah-Alava, A. (1960). Genetic mosaics. Sci. Am. 202 #5, 118–130. 1716. Hannah-Alava, A. and Stern, C. (1957). The sexcombs in

354

1717.

1718. 1719.

1720.

1721. 1722.

1723. 1724.

1725.

1726.

1727.

1728.

1729. 1730. 1731.

1732.

1733.

1734.

1735.

1736.

REFERENCES

males and intersexes of Drosophila melanogaster. J. Exp. Zool. 134, 533–556. Harbecke, R., Meise, M., Holz, A., Klapper, R., Nafﬁn, E., Nordhoff, V., and Janning, W. (1996). Larval and imaginal pathways in early development of Drosophila. Int. J. Dev. Biol. 40, 197–204. Harbury, P.B., Kim, P.S., and Alber, T. (1994). Crystal structure of an isoleucine-zipper trimer. Nature 371, 80–83. Harbury, P.B., Zhang, T., Kim, P.S., and Alber, T. (1993). A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science 262, 1401– 1407. Hardie, R.C. (1985). Functional organization of the ﬂy retina. In “Progress in Sensory Physiology,” Vol. 5 (D. Ottoson, ed.). Springer-Verlag: Berlin, pp. 1–79. Hardie, R.C. (1986). The photoreceptor array of the dipteran retina. Trends Neurosci. 9, 419–423. Harding, K., Rushlow, C., Doyle, H.J., Hoey, T., and Levine, M. (1986). Cross-regulatory interactions among pair-rule genes in Drosophila. Science 233, 953–959. Hardy, J. and Isra¨el, A. (1999). In search of γ-secretase. Nature 398, 466–467. Hariharan, I.K., Carthew, R.W., and Rubin, G.M. (1991). The Drosophila Roughened mutation: activation of a rap homolog disrupts eye development and interferes with cell determination. Cell 67, 717–722. Harrelson, A.L. and Goodman, C.S. (1988). Growth cone guidance in insects: fasciclin II is a member of the immunoglobulin superfamily. Science 242, 700–708. Harris, W.A., Stark, W.S., and Walker, J.A. (1976). Genetic dissection of the photoreceptor system in the compound eye of Drosophila melanogaster. J. Physiol. 256, 415–439. Harrison, L.G. (1993). “Kinetic Theory of Living Pattern.” Developmental and Cell Biology Series, Vol. 28. Cambridge Univ. Pr., Cambridge. Harrison, L.G. and Tan, K.Y. (1988). Where may reactiondiffusion mechanisms be operating in metameric patterning of Drosophila embryos? BioEssays 8, 118–124. Harrison, S.C. (1991). A structural taxonomy of DNAbinding domains. Nature 353, 715–719. Harrison, S.C. (1996). Peptide-surface association: the case of PDZ and PTB domains. Cell 86, 341–343. Harrison, S.D. and Travers, A.A. (1990). The tramtrack gene encodes a Drosophila ﬁnger protein that interacts with the ftz transcriptional regulatory region and shows a novel embryonic expression pattern. EMBO J. 9, 207– 216. Hart, A.C., Kr¨amer, H., Van Vactor, D.L., Jr., Paidhungat, M., and Zipursky, S.L. (1990). Induction of cell fate in the Drosophila retina: the bride of sevenless protein is predicted to contain a large extracellular domain and seven transmembrane segments. Genes Dev. 4, 1835–1847. Hart, A.C., Kr¨amer, H., and Zipursky, S.L. (1993). Extracellular domain of the boss transmembrane ligand acts as an antagonist of the sev receptor. Nature 361, 732–736. Hart, C.M., Ketel, C.S., and Simon, J.A. (2001). Characterization of the ESC-E(Z) complex and functional partnership. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a95. Hart, K. and Bienz, M. (1996). A test for cell autonomy, based on di-cistronic messenger translation. Development 122, 747–751. Hart, M., Concordet, J.-P., Lassot, I., Albert, I., del los Santos, R., Durand, H., Perret, C., Rubinfeld, B., Margottin,

1737.

1738.

1739. 1740.

1741.

1742.

1743.

1744.

1745.

1746.

1747.

1748.

1749.

1750.

1751.

1752.

F., Benarous, R., and Polakis, P. (1999). The F-box protein β-TrCP associates with phosphorylated β-catenin and regulates its activity in the cell. Curr. Biol. 9, 207– 210. Hartenstein, A.Y., Rugendorff, A., Tepass, U., and Hartenstein, V. (1992). The function of the neurogenic genes during epithelial development in the Drosophila embryo. Development 116, 1203–1220. Hartenstein, K., Sinha, P., Mishra, A., Schenkel, H., T¨or¨ok, I., and Mechler, B.M. (1997). The congested-like tracheae gene of Drosophila melanogaster encodes a member of the mitochondrial carrier family required for gas-ﬁlling of the tracheal system and expansion of the wings after eclosion. Genetics 147, 1755–1768. Hartenstein, V. (1993). “Atlas of Drosophila Development.” Cold Spring Harbor, N.Y. Hartenstein, V. and Campos-Ortega, J.A. (1984). Early neurogenesis in wild-type Drosophila melanogaster. Roux’s Arch. Dev. Biol. 193, 308–325. Hartenstein, V. and Posakony, J.W. (1989). Development of adult sensilla on the wing and notum of Drosophila melanogaster. Development 107, 389–405. Hartenstein, V. and Posakony, J.W. (1990). A dual function of the Notch gene in Drosophila sensillum development. Dev. Biol. 142, 13–30. Hartenstein, V. and Posakony, J.W. (1990). Sensillum development in the absence of cell division: the sensillum phenotype of the Drosophila mutant string. Dev. Biol. 138, 147–158. Hartenstein, V., Tepass, U., and Gruszynski-deFeo, E. (1996). Proneural and neurogenic genes control speciﬁcation and morphogenesis of stomatogastric nerve cell precursors in Drosophila. Dev. Biol. 173, 213–227. Hartenstein, V., Younossi-Hartenstein, A., and Lekven, A. (1994). Delamination and division in the Drosophila neurectoderm: spatiotemporal pattern, cytoskeletal dynamics, and common control by neurogenic and segment polarity genes. Dev. Biol. 165, 480–499. Hartley, D.A., Preiss, A., and Artavanis-Tsakonas, S. (1988). A deduced gene product from the Drosophila neurogenic locus, Enhancer of Split, shows homology to mammalian G-protein β subunit. Cell 55, 785–795. Hartley, D.A., Xu, T., and Artavanis-Tsakonas, S. (1987). The embryonic expression of the Notch locus of Drosophila melanogaster and the implications of point mutations in the extracellular EGF-like domain of the predicted protein. EMBO J. 6, 3407–3417. Hartman, J.L., IV, Garvik, B., and Hartwell, L. (2001). Principles for the buffering of genetic variation. Science 291, 1001–1004. Hartmann, B., Marty, T., and Affolter, M. (2001). Functional analysis of the Drosophila Schnurri protein in vivo. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a162. Hartmann, C., Taubert, H., J¨ackle, H., and Pankratz, M.J. (1994). A two-step mode of stripe formation in the Drosophila blastoderm requires interactions among primary pair rule genes. Mechs. Dev. 45, 3–13. Haskell, R.E. (1993). “Introduction to Computer Engineering: Logic Design and the 8086 Microprocessor.” PrenticeHall, Englewood Cliffs, N. J. Hassan, A.H., Neely, K.E., and Workman, J.L. (2001). Histone acetyltransferase complexes stabilize SWI/SNF binding to promoter nucleosomes. Cell 104, 817–827.

REFERENCES

1753. Hassan, B., Li, L., Bremer, K.A., Chang, W., Pinsonneault, J., and Vaessin, H. (1997). Prospero is a panneural transcription factor that modulates homeodomain protein activity. Proc. Natl. Acad. Sci. USA 94, 10991–10996. 1754. Hassan, B. and Vaessin, H. (1996). Regulatory interactions during early neurogenesis in Drosophila. Dev. Genet. 18, 18–27. 1755. Hassan, B. and Vaessin, H. (1997). Daughterless is required for the expression of cell cycle genes in peripheral nervous system precursors of Drosophila embryos. Dev. Genet. 21, 117–122. 1756. Hassan, B.A. and Bellen, H.J. (2000). Doing the MATH: is the mouse a good model for ﬂy development? Genes Dev. 14, 1852–1865. 1757. Hastie, N.D. (1991). Pax in our time. Curr. Biol. 1, 342–344. 1758. Hasty, J., McMillen, D., Isaacs, F., and Collins, J.J. (2001). Computational studies of gene regulatory networks: in numero molecular biology. Nature Rev. Genet. 2, 268– 279. 1759. Hatini, V., Bokor, P., Goto-Mandeville, R., and DiNardo, S. (2000). Tissue and stage-speciﬁc modulation of Wingless signaling by the segment polarity gene lines. Genes Dev. 14, 1364–1376. 1760. Hauck, B., Gehring, W.J., and Walldorf, U. (1999). Functional analysis of an eye speciﬁc enhancer of the eyeless gene in Drosophila. Proc. Natl. Acad. Sci. USA 96, 564–569. 1761. Hawkins, N. and Garriga, G. (1998). Asymmetric cell division: from A to Z. Genes Dev. 12, 3625–3638. ¨ 1762. Hawkins, N.C., Thorpe, J., and Schupbach, T. (1996). encore, a gene required for the regulation of germ line mitosis and oocyte differentiation during Drosophila oogenesis. Development 122, 281–290. 1763. Hay, B.A., Wolff, T., and Rubin, G.M. (1994). Expression of baculovirus P35 prevents cell death in Drosophila. Development 120, 2121–2129. 1764. Hayashi, S. (1996). A Cdc2 dependent checkpoint maintains diploidy in Drosophila. Development 122, 1051– 1058. 1765. Hayashi, S., Hirose, S., Metcalfe, T., and Shirras, A.D. (1993). Control of imaginal cell development by the escargot gene of Drosophila. Development 118, 105–115. 1766. Hayashi, S., Rubinfeld, B., Souza, B., Polakis, P., and Wieschaus, E. (1997). A Drosophila homolog of the tumor suppressor gene adenomatous polyposis coli down-regulates β-catenin but its zygotic expression is not essential for the regulation of Armadillo. Proc. Natl. Acad. Sci. USA 94, 242–247. 1767. Hayashi, S. and Scott, M.P. (1990). What determines the speciﬁcity of action of Drosophila homeodomain proteins? Cell 63, 883–894. 1768. Hayashi, T., Kojima, T., and Saigo, K. (1998). Speciﬁcation of primary pigment cell and outer photoreceptor fates by BarH1 homeobox gene in the developing Drosophila eye. Dev. Biol. 200, 131–145. 1769. Hayes, B. (1999). E pluribus unum. Am. Sci. 87, 10–14. 1770. Hayes, J.J. and Hansen, J.C. (2001). Nucleosomes and the chromosome ﬁber. Curr. Opin. Gen. Dev. 11, 124–129. 1771. Hayes, P.H. (1982). “Mutant Analysis of Determination in Drosophila.” Ph.D. dissertation, Dept. of Genetics, Univ. of Alberta, Edmonton, Alberta, Canada. 1772. Hayes, P.H., Sato, T., and Denell, R.E. (1984). Homoeosis in Drosophila: the Ultrabithorax larval syndrome. Proc. Natl. Acad. Sci. USA 81, 545–549. 1773. Haynie, J. and Schubiger, G. (1979). Absence of distal to

355

1774.

1775.

1776.

1777.

1778.

1779.

1780.

1781.

1782.

1783.

1784.

1785.

1786.

1787.

1788.

1789.

1790.

1791.

proximal intercalary regeneration in imaginal wing discs of Drosophila melanogaster. Dev. Biol. 68, 151–161. Haynie, J.L. (1982). Homologies of positional information in thoracic imaginal discs of Drosophila melanogaster. W. Roux’s Archiv. 191, 293–300. Haynie, J.L. and Bryant, P.J. (1976). Intercalary regeneration in imaginal wing disk of Drosophila melanogaster. Nature 259, 659–662. Haynie, J.L. and Bryant, P.J. (1977). The effects of X-rays on the proliferation dynamics of cells in the imaginal wing disc of Drosophila melanogaster. W. Roux’s Arch. 183, 85– 100. Haynie, J.L. and Bryant, P.J. (1986). Development of the eye-antenna imaginal disc and morphogenesis of the adult head in Drosophila melanogaster. J. Exp. Zool. 237, 293–308. Hays, R., Buchanan, K.T., Neff, C., and Orenic, T.V. (1999). Patterning of Drosophila leg sensory organs through combinatorial signaling by Hedgehog, Decapentaplegic and Wingless. Development 126, 2891–2899. Hays, R., Gibori, G.B., and Bejsovec, A. (1997). Wingless signaling generates pattern through two distinct mechanisms. Development 124, 3727–3736. Hazelett, D.J., Bourouis, M., Walldorf, U., and Treisman, J.E. (1998). decapentaplegic and wingless are regulated by eyes absent and eyegone and interact to direct the pattern of retinal differentiation in the eye disc. Development 125, 3741–3751. Heberlein, U., Borod, E.R., and Chanut, F.A. (1998). Dorsoventral patterning in the Drosophila retina by wingless. Development 125, 567–577. Heberlein, U., Hariharan, I.K., and Rubin, G.M. (1993). Star is required for neuronal differentiation in the Drosophila retina and displays dosage-sensitive interactions with Ras1. Dev. Biol. 160, 51–63. Heberlein, U., Mlodzik, M., and Rubin, G.M. (1991). Cellfate determination in the developing Drosophila eye: role of the rough gene. Development 112, 703–712. Heberlein, U. and Moses, K. (1995). Mechanisms of Drosophila retinal morphogenesis: the virtues of being progressive. Cell 81, 987–990. Heberlein, U. and Rubin, G.M. (1991). Star is required in a subset of photoreceptor cells in the developing Drosophila retina and displays dosage sensitive interactions with rough. Dev. Biol. 144, 353–361. Heberlein, U., Singh, C.M., Luk, A.Y., and Donohoe, T.J. (1995). Growth and differentiation in the Drosophila eye coordinated by hedgehog. Nature 373, 709–711. Heberlein, U. and Treisman, J.E. (2000). Early retinal development in Drosophila. In “Vertebrate Eye Development,” Results and Problems in Cell Differentiation, Vol. 31 (M.E. Fini, ed.). Springer: Berlin, pp. 37–50. Heberlein, U., Wolff, T., and Rubin, G.M. (1993). The TGFβ homolog dpp and the segment polarity gene hedgehog are required for propagation of a morphogenetic wave in the Drosophila retina. Cell 75, 913–926. Heemskerk, J. and DiNardo, S. (1994). Drosophila hedgehog acts as a morphogen in cellular patterning. Cell 76, 449–460. Heemskerk, J., DiNardo, S., Kostriken, R., and O’Farrell, P.H. (1991). Multiple modes of engrailed regulation in the progression towards cell fate determination. Nature 352, 404–410. Heilig, J.S., Freeman, M., Laverty, T., Lee, K.J., Campos,

356

1792.

1793.

1794.

1795.

1796.

1797. 1798.

1799.

1800.

1801.

1802.

1803.

1804. 1805.

1806.

1807. 1808.

1809.

REFERENCES

A.R., Rubin, G.M., and Steller, H. (1991). Isolation and characterization of the disconnected gene of Drosophila melanogaster. EMBO J. 10, 809–815. Heintzen, C., Loros, J.J., and Dunlap, J.C. (2001). The PAS protein VIVID deﬁnes a clock-associated feedback loop that represses light input, modulates gating, and regulates clock resetting. Cell 104, 453–464. Heitz, F., Morris, M.C., Fesquet, D., Cavadore, J.-C., Dor´ee, M., and Divita, G. (1997). Interactions of cyclins with cyclin-dependent kinases: a common interactive mechanism. Biochemistry 36, 4995–5003. Heitzler, P., Bourouis, M., Ruel, L., Carteret, C., and Simpson, P. (1996). Genes of the Enhancer of split and achaete-scute complexes are required for a regulatory loop between Notch and Delta during lateral signalling in Drosophila. Development 122, 161–171. Heitzler, P., Coulson, D., Saenz-Robles, M.-T., Ashburner, M., Roote, J., Simpson, P., and Gubb, D. (1993). Genetic and cytogenetic analysis of the 43A-E region containing the segment polarity gene costa and the cellular polarity genes prickle and spiny-legs in Drosophila melanogaster. Genetics 135, 105–115. Heitzler, P., Haenlin, M., Ramain, P., Calleja, M., and Simpson, P. (1996). A genetic analysis of pannier, a gene necessary for viability of dorsal tissues and bristle positioning in Drosophila. Genetics 143, 1271–1286. Heitzler, P. and Simpson, P. (1991). The choice of cell fate in the epidermis of Drosophila. Cell 64, 1083–1092. Heitzler, P. and Simpson, P. (1993). Altered epidermal growth factor-like sequences provide evidence for a role of Notch as a receptor in cell fate decisions. Development 117, 1113–1123. Held, L.I., Jr. (1977). “Analysis of Bristle-Pattern Formation in Drosophila.” Ph.D. dissertation, Dept. of Molecular Biology, Univ. of California, Berkeley. Held, L.I., Jr. (1979). A high-resolution morphogenetic map of the second-leg basitarsus in Drosophila melanogaster. Wilhelm Roux’s Arch. 187, 129–150. Held, L.I., Jr. (1979). Pattern as a function of cell number and cell size on the second-leg basitarsus of Drosophila. Wilhelm Roux’s Arch. 187, 105–127. Held, L.I., Jr. (1990). Arrangement of bristles as a function of bristle number on a leg segment in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 199, 48–62. Held, L.I., Jr. (1990). Sensitive periods for abnormal patterning on a leg segment in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 199, 31–47. Held, L.I., Jr. (1991). Bristle patterning in Drosophila. BioEssays 13, 633–640. Held, L.I., Jr. (1992). “Models for Embryonic Periodicity.” Monographs in Developmental Biology, Vol. 24. Karger, Basel. Held, L.I., Jr. (1993). Segment-polarity mutations cause stripes of defects along a leg segment in Drosophila. Dev. Biol. 157, 240–250. Held, L.I., Jr. (1995). Axes, boundaries and coordinates: the ABCs of ﬂy leg development. BioEssays 17, 721–732. Held, L.I., Jr. and Bryant, P.J. (1984). Cell interactions controlling the formation of bristle patterns in Drosophila. In “Pattern Formation: A Primer in Developmental Biology,” (G.M. Malacinski and S.V. Bryant, eds.). Macmillan: New York, pp. 291–322. Held, L.I., Jr., Duarte, C.M., and Derakhshanian, K. (1986). Extra joints and misoriented bristles on Drosophila legs. In

1810.

1811.

1812.

1813.

1814. 1815.

1816.

1817.

1818.

1819. 1820.

1821.

1822.

1823. 1824.

1825. 1826.

1827.

1828.

“Progress in Developmental Biology (Part A),” (H. Slavkin, ed.). Alan R. Liss: N. Y., pp. 293–296. Held, L.I., Jr., Duarte, C.M., and Derakhshanian, K. (1986). Extra tarsal joints and abnormal cuticular polarities in various mutants of Drosophila melanogaster. Roux’s Arch. Dev. Biol. 195, 145–157. Held, L.I., Jr. and Heup, M. (1996). Genetic mosaic analysis of decapentaplegic and wingless gene function in the Drosophila leg. Dev. Genes Evol. 206, 180–194. Held, L.I., Jr., Heup, M.A., Sappington, J.M., and Peters, S.D. (1994). Interactions of decapentaplegic, wingless, and Distal-less in the Drosophila leg. Roux’s Arch. Dev. Biol. 203, 310–319. Held, L.I., Jr. and Pham, T.T. (1983). Accuracy of bristle placement on a leg segment in Drosophila melanogaster. J. Morph. 178, 105–110. Heldin, C.-H. (1995). Dimerization of cell surface receptors in signal transduction. Cell 80, 213–223. Heldin, C.-H., Miyazono, K., and ten Dijke, P. (1997). TGF-β signalling from cell membrane to nucleus through SMAD proteins. Nature 390, 465–471. Helms, W., Lee, H., Ammerman, M., Parks, A.L., Muskavitch, M.A.T., and Yedvobnick, B. (1999). Engineered truncations in the Drosophila Mastermind protein disrupt Notch pathway function. Dev. Biol. 215, 358–374. Helps, N.R., Cohen, P.T.W., Bahri, S.M., Chia, W., and Babu, K. (2001). Interaction with protein phosphatase 1 is essential for bifocal function during the morphogenesis of the Drosophila compound eye. Mol. Cell. Biol. 21, 2154–2164. Hemmati-Brivanlou, A. and Melton, D. (1997). Vertebrate embryonic cells will become nerve cells unless told otherwise. Cell 88, 13–17. Hemmings, B.A. (1997). PH domains–a universal membrane adapter. Science 275, 1899. Henderson, K.D., Isaac, D.D., and Andrew, D.J. (1999). Cell fate speciﬁcation in the Drosophila salivary gland: the integration of homeotic gene function with the DPP signaling cascade. Dev. Biol. 205, 10–21. Hendrickson, J.E. and Sakonju, S. (1995). Cis and trans interactions between the iab regulatory regions and abdominal-A and Abdominal-B in Drosophila melanogaster. Genetics 139, 835–848. Henery, C.C., Bard, J.B.L., and Kaufman, M.H. (1992). Tetraploidy in mice, embryonic cell number, and the grain of the developmental map. Dev. Biol. 152, 233–241. Henikoff, S. (1998). Conspiracy of silence among repeated transgenes. BioEssays 20, 532–535. Henikoff, S., Greene, E.A., Pietrokovski, S., Bork, P., Attwood, T.K., and Hood, L. (1997). Gene families: the taxonomy of protein paralogs and chimeras. Science 278, 609–614. Henikoff, S. and Matzke, M.A. (1997). Exploring and explaining epigenetic effects. Trends Genet. 13, 293–295. ¨ Henke, K. and Maas, H. (1946). Uber sensible Perioden der allgemeinen K¨orpergliederung von Drosophila. Nachr. Akad. Wiss. G¨ottingen. Math.-Phys. Kl. IIb, Biol.-Physiol.Chem. Abt. 1, 3–4. Hepker, J., Blackman, R.K., and Holmgren, R. (1999). Cubitus interruptus is necessary but not sufﬁcient for direct activation of a wing-speciﬁc decapentaplegic enhancer. Development 126, 3669–3677. Hepker, J., Wang, Q.-T., Motzny, C.K., Holmgren, R., and Orenic, T.V. (1997). Drosophila cubitus interruptus forms a negative feedback loop with patched and regulates

REFERENCES

1829.

1830.

1831.

1832.

1833.

1834.

1835.

1836.

1837.

1838. 1839. 1840. 1841.

1842.

1843.

1844.

1845.

1846.

expression of Hedgehog target genes. Development 124, 549–558. Herbst, R., Carroll, P.M., Allard, J.D., Schilling, J., Raabe, T., and Simon, M.A. (1996). Daughter of sevenless is a substrate of the phosphotyrosine phosphatase Corkscrew and functions during Sevenless signaling. Cell 85, 899–909. Herbst, R., Zhang, X., Qin, J., and Simon, M.A. (1999). Recruitment of the protein tyrosine phosphatase CSW by DOS is an essential step during signaling by the Sevenless receptor tyrosine kinase. EMBO J. 18, 6950–6961. H´erich´e, J.-K. and O’Farrell, P.H. (2001). Genetic analysis of Cul-1 in Drosophila. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a136. Herr, W. and Cleary, M.A. (1995). The POU domain: versatility in transcriptional regulation by a ﬂexible two-in-one DNA-binding domain. Genes Dev. 9, 1679–1693. Heslip, T.R., Theisen, H., Walker, H., and Marsh, J.L. (1997). SHAGGY and DISHEVELLED exert opposite effects on wingless and decapentaplegic expression and on positional identity in imaginal discs. Development 124, 1069– 1078. Heuer, J.G. and Kaufman, T.C. (1992). Homeotic genes have speciﬁc functional roles in the establishment of the Drosophila embryonic peripheral nervous system. Development 115, 35–47. Hewitt, G.F., Strunk, B.S., Margulies, C., Priputin, T., Wang, X.-D., Amey, R., Pabst, B.A., Kosman, D., Reinitz, J., and Arnosti, D.N. (1999). Transcriptional repression by the Drosophila Giant protein: cis element positioning provides an alternative means of interpreting an effector gradient. Development 126, 1201–1210. Hidalgo, A. (1991). Interactions between segment polarity genes and the generation of the segmental pattern in Drosophila. Mechs. Dev. 35, 77–87. Hidalgo, A. (1994). Three distinct roles for the engrailed gene in Drosophila wing development. Curr. Biol. 4, 1087– 1098. Hidalgo, A. (1996). The roles of engrailed. Trends Genet. 12, 1–4. Hidalgo, A. (1998). Growth and patterning from the engrailed interface. Int. J. Dev. Biol. 42, 317–324. Hidalgo, A. (1998). Wing patterning knot untangled. BioEssays 20, 449–452. Hidalgo, A. and Ingham, P. (1990). Cell patterning in the Drosophila segment: spatial regulation of the segment polarity gene patched. Development 110, 291–301. Higashijima, S.-i., Kojima, T., Michiue, T., Ishimaru, S., Emori, Y., and Saigo, K. (1992). Dual Bar homeo box genes of Drosophila required in two photoreceptor cells, R1 and R6, and primary pigment cells for normal eye development. Genes Dev. 6, 50–60. Higashijima, S.-i., Michiue, T., Emori, Y., and Saigo, K. (1992). Subtype determination of Drosophila embryonic external sensory organs by redundant homeo box genes BarH1 and BarH2. Genes Dev. 6, 1005–1018. High, S. and Dobberstein, B. (1992). Mechanisms that determine the transmembrane disposition of proteins. Curr. Opin. Cell Biol. 4, 581–586. Hildreth, P.E. (1965). Doublesex, a recessive gene that transforms both males and females of Drosophila into intersexes. Genetics 51, 659–678. Hill, C.S. and Treisman, R. (1995). Transcriptional regulation by extracellular signals: mechanisms and speciﬁcity. Cell 80, 199–211.

357

1847. Hill, R. and Van Heyningen, V. (1992). Mouse mutations and human disorders are Paired. Trends Genet. 8, 119–120. 1848. Hill, R.E. and Davidson, D.R. (1994). Seeing eye to eye. Curr. Biol. 4, 1155–1157. 1849. Hillier, B.J., Christopherson, K.S., Prehoda, K.E., Bredt, D.S., and Lim, W.A. (1999). Unexpected modes of PDZ domain scaffolding revealed by structure of nNOSsyntrophin complex. Science 284, 812–815. 1850. Hing, H., Xiao, J., Harden, N., Lim, L., and Zipursky, S.L. (1999). Pak functions downstream of Dock to regulate photoreceptor axon guidance in Drosophila. Cell 97, 853– 863. 1851. Hing, H.K., Sun, X., and Artavanis-Tsakonas, S. (1994). Modulation of wingless signaling by Notch in Drosophila. Mechs. Dev. 47, 261–268. 1852. Hinshaw, J.E. and Schmid, S.L. (1995). Dynamin selfassembles into rings suggesting a mechanism for coated vesicle budding. Nature 374, 190–192. 1853. Hinton, H.E. (1970). Some little known surface structures. In “Insect Ultrastructure,” 5th Symp. Roy. Entomol. Soc. Lond., (A.C. Neville, ed.). Blackwell: Oxford, pp. 41–58. 1854. Hinz, U., Giebel, B., and Campos-Ortega, J.A. (1994). The basic-helix-loop-helix domain of Drosophila lethal of scute protein is sufﬁcient for proneural function and activates neurogenic genes. Cell 76, 77–87. 1855. Hirata, J., Nakagoshi, H., Nabeshima, Y.-i., and Matsuzaki, F. (1995). Asymmetric segregation of the homeodomain protein Prospero during Drosophila development. Nature 377, 627–630. 1856. Hiromi, Y., Mlodzik, M., West, S.R., Rubin, G.M., and Goodman, C.S. (1993). Ectopic expression of seven-up causes cell fate changes during ommatidial assembly. Development 118, 1123–1135. 1857. Hirota, Y., Okabe, M., Imai, T., Kurusu, M., Yamamoto, A., Miyao, S., Nakamura, M., Sawamoto, K., and Okano, H. (1999). Musashi and Seven in absentia downregulate Tramtrack through distinct mechanisms in Drosophila eye development. Mechs. Dev. 87, 93–101. 1858. Hirsch, J.A. and Aggarwal, A.K. (1995). Structure of the Even-skipped homeodomain complexed to AT-rich DNA: new perspectives on homeodomain speciﬁcity. EMBO J. 14, 6280–6291. 1859. Ho, M.-W., Bolton, E., and Saunders, P.T. (1983). Bithorax phenocopy and pattern formation. I. Spatiotemporal characteristics of the phenocopy response. Exp. Cell Biol. 51, 282–290. 1860. Ho, M.-W., Saunders, P.T., and Bolton, E. (1983). Bithorax phenocopy and pattern formation. II. A model of prepattern formation. Exp. Cell Biol. 51, 291–299. 1861. Hobert, O. and Ruvkun, G. (1999). Pax genes in Caenorhabditis elegans: a new twist. Trends Genet. 15, 214–216. 1862. Hobert, O. and Westphal, H. (2000). Functions of LIMhomeobox genes. Trends Genet. 16, 75–83. 1863. Hodges, A. (1983). “Alan Turing: The Enigma.” Simon & Schuster, New York. 1864. Hodges, R.S. (1992). Unzipping the secrets of coiled-coils. Curr. Biol. 2, 122–124. 1865. Hodgkin, J. (1998). Seven types of pleiotropy. Int. J. Dev. Biol. 42, 501–505. 1866. Hodgkin, N.M. and Bryant, P.J. (1978). Scanning electron microscopy of the adult of Drosophila melanogaster. In “The Genetics and Biology of Drosophila,” Vol. 2c (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 337–358.

358

1867. Hodgson, J.W., Cheng, N.N., Sinclair, D.A.R., Kyba, M., Randsholt, N.B., and Brock, H.W. (1997). The polyhomeotic locus of Drosophila melanogaster is transcriptionally and post-transcriptionally regulated during embryogenesis. Mechs. Dev. 66, 69–81. 1868. Hoey, T. and Levine, M. (1988). Divergent homeo box proteins recognize similar DNA sequences in Drosophila. Nature 332, 858–861. 1869. Hofbauer, A. and Campos-Ortega, J.A. (1976). Cell clones and pattern formation: genetic eye mosaics in Drosophila melanogaster. W. Roux’s Arch. 179, 275–289. 1870. Hoffman, M. (1991). New role found for a common protein “motif”. Science 253, 742. 1871. Hoffmann, F.M. (1990). Developmental functions of decapentaplegic, a member of the TGF-β family in Drosophila. In “Genetics of Pattern Formation and Growth Control,” Symp. Soc. Dev. Biol., Vol. 48 (A.P. Mahowald, ed.). Wiley-Liss: New York, pp. 103–124. 1872. Hoffmann, F.M. and Goodman, W. (1987). Identiﬁcation in transgenic animals of the Drosophila decapentaplegic sequences required for embryonic dorsal pattern formation. Genes Dev. 1, 615–625. 1873. Hofstadter, D.R. (1979). “G¨odel, Escher, Bach: An Eternal Golden Braid.” Random House, New York. 1874. Hogan, B.L.M. (1994). Sorting out the signals. Curr. Biol. 4, 1122–1124. 1875. Hoge, M. (1915). The inﬂuence of temperature on the development of a Mendelian character. J. Exp. Zool. 18, 241– 297. 1876. Holland, P.W.H. (1999). The future of evolutionary developmental biology. Nature 402 Suppl., C41–C44. 1877. Holland, P.W.H. (1999). Gene duplication: past, present and future. Sems. Cell Dev. Biol. 10, 541–547. ¨ 1878. Holley, S.A., Geisler, R., and Nusslein-Volhard, C. (2000). Control of her1 expression during zebraﬁsh somitogenesis by a Delta-dependent oscillator and an independent wave-front activity. Genes Dev. 14, 1678–1690. 1879. Holliday, R. (1988). Successes and limitations of molecular biology. J. Theor. Biol. 132, 253–262. 1880. Holliday, R. (1990). Mechanisms for the control of gene activity during development. Biol. Rev. 65, 431–471. 1881. Holliday, R. (1990). Paradoxes between genetics and development. J. Cell Sci. 97, 395–398. 1882. Hollingsworth, M.J. (1960). The morphology of intersexes in Drosophila subobscura. J. Exp. Zool. 143, 123–151. 1883. Hollingsworth, M.J. (1964). Sex-combs of intersexes and the arrangement of the chaetae on the legs of Drosophila. J. Morph. 115, 35–51. 1884. Holmes, F.L. (2000). Seymour Benzer and the deﬁnition of the gene. In “The Concept of the Gene in Development and Evolution: Historical and Epistemological Perspectives,” (P.J. Beurton, R. Falk, and H.-J. Rheinberger, eds.). Cambridge Univ. Pr.: Cambridge, pp. 115–155. 1885. Holtfreter, J. (1951). Some aspects of embryonic induction. Growth 15 (Suppl.), 117–152. 1886. Holz, A., Meise, M., and Janning, W. (1997). Adepithelial cells in Drosophila melanogaster: origin and cell lineage. Mechs. Dev. 62, 93–101. 1887. Homyk, T., Jr., Isono, K., and Pak, W.L. (1985). Developmental and physiological analysis of a conditional mutation affecting photoreceptor and optic lobe development in Drosophila melanogaster. J. Neurogenet. 2, 309–324. 1888. Honda, H., Kodama, R., Takeuchi, T., Yamanaka, H., Watanabe, K., and Eguchi, G. (1984). Cell behaviour in a

REFERENCES

1889.

1890.

1891.

1892.

1893.

1894.

1895.

1896. 1897. 1898.

1899.

1900.

1901.

1902.

1903.

1904.

1905.

1906. 1907.

1908.

polygonal cell sheet. J. Embyrol. Exp. Morph. 83 (Suppl.), 313–327. Honda, H., Tanemura, M., and Yoshida, A. (1990). Estimation of neuroblast numbers in insect neurogenesis using the lateral inhibition hypothesis of cell differentiation. Development 110, 1349–1352. Honda, H. and Yamanaka, H. (1984). A computer simulation of geometrical conﬁgurations during cell division. J. Theor. Biol. 106, 423–435. Honjo, T. (1996). The shortest path from the surface to the nucleus: RBP-Jκ/Su(H) transcription factor. Genes to Cells 1, 1–9. Honma, T. and Goto, K. (2001). Complexes of MADS-box proteins are sufﬁcient to convert leaves into ﬂoral organs. Nature 409, 525–529. Hooper, J. (2001). Modulation of Hedgehog signaling by heterotrimeric G proteins. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a334. Hooper, J.E. (1994). Distinct pathways for autocrine and paracrine Wingless signalling in Drosophila embryos. Nature 372, 461–464. Hooper, J.E. and Scott, M.P. (1989). The Drosophila patched gene encodes a putative membrane protein required for segmental patterning. Cell 59, 751–765. Hooper, N.M. (1998). Membrane biology: Do glycolipid microdomains really exist? Curr. Biol. 8, R114–R116. Hopcroft, J.E. (1984). Turing machines. Sci. Am. 250 #5, 86–98. Hopmann, R. and Miller, K. (2001). Bristle growth in the absence of actin bundles. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a76. [Cf. 2002 Cell 108, 233–246.] Hoppe, P.E. and Greenspan, R.J. (1986). Local function of the Notch gene for embryonic ectodermal pathway choice in Drosophila. Cell 46, 773–783. Hoppe, P.E. and Greenspan, R.J. (1990). The Notch locus of Drosophila is required in epidermal cells for epidermal development. Development 109, 875–885. Hoppler, S. and Bienz, M. (1994). Speciﬁcation of a single cell type by a Drosophila homeotic gene. Cell 76, 689– 702. Horard, B., Tatout, C., Poux, S., and Pirrotta, V. (2000). Structure of a Polycomb response element and in vitro binding of Polycomb group complexes containing GAGA factor. Mol. Cell. Biol. 20, 3187–3197. Horsﬁeld, J., Penton, A., Secombe, J., Hoffmann, F.M., and Richardson, H. (1998). decapentaplegic is required for arrest in G1 phase during Drosophila eye development. Development 125, 5069–5078. Horvitz, H.R. and Herskowitz, I. (1992). Mechanisms of asymmetric cell division: Two Bs or not two Bs, that is the question. Cell 68, 237–255. Hoschuetzky, H., Aberle, H., and Kemler, R. (1994). β-catenin mediates the interaction of the cadherin-catenin complex with Epidermal Growth Factor Receptor. J. Cell Biol. 127, 1375–1380. Hou, X.S. and Perrimon, N. (1997). The JAK-STAT pathway in Drosophila. Trends Genet. 13, 105–110. Hough, C.D., Woods, D.F., Park, S., and Bryant, P.J. (1997). Organizing a functional junctional complex requires speciﬁc domains of the Drosophila MAGUK Discs large. Genes Dev. 11, 3242–3253. Howard, K.R. and Struhl, G. (1990). Decoding positional information: regulation of the pair-rule gene hairy. Development 110, 1223–1231.

REFERENCES

1909. Howells, A.J. (1972). Levels of RNA and DNA in Drosophila melanogaster at different stages of development: a comparison between one bobbed and two phenotypically non-bobbed stocks. Biochem. Genet. 6, 217–230. 1910. Howes, R. and Bray, S. (2000). Pattern formation: Wingless on the move. Curr. Biol. 10, R222–R226. 1911. Howes, R., Wasserman, J.D., and Freeman, M. (1998). In vivo analysis of Argos structure-function. J. Biol. Chem. 273 #7, 4275–4281. 1912. Hoyle, H.D., Turner, F.R., and Raff, E.C. (2000). A transient specialization of the microtubule cytoskeleton is required for differentiation of the Drosophila visual system. Dev. Biol. 221, 375–389. 1913. Hsieh, J.J.-D., Zhou, S., Chen, L., Young, D.B., and Hayward, S.D. (1999). CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex. Proc. Natl. Acad. Sci. USA 96, 23–28. 1914. Hsieh, J.J.D., Henkel, T., Salmon, P., Robey, E., Peterson, M.G., and Hayward, S.D. (1996). Truncated mammalian Notch1 activates CBF1/RBPJκ-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2. Mol. Cell. Biol. 16, 952–959. 1915. Hsiung, F. and Moses, K. (2001). Star mediates Spi maturation via direct physical interaction. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a170. [See also 2001 Mechs. Dev. 107, 13–23.] 1916. Hsu, J.-C. and Perrimon, N. (1994). A temperaturesensitive MEK mutation demonstrates the conservation of the signaling pathways activated by receptor tyrosine kinases. Genes Dev. 8, 2176–2187. 1917. Hsu, S.-C., Galceran, J., and Grosschedl, R. (1998). Modulation of transcriptional regulation by LEF-1 in response to Wnt-1 signaling and association with β-catenin. Mol. Cell. Biol. 18, 4807–4818. 1918. Hsu, T., Gogos, J.A., Kirsh, S.A., and Kafatos, F.C. (1992). Multiple zinc ﬁnger forms resulting from developmentally regulated alternative splicing of a transcription factor gene. Science 257, 1946–1950. 1919. Hsu, W., Zeng, L., and Costantini, F. (1999). Identiﬁcation of a domain of Axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain. J. Biol. Chem. 274 #6, 3439–3445. 1920. Hsueh, Y.-P., Wang, T.-F., Yang, F.-C., and Sheng, M. (2000). Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2. Nature 404, 298–302. 1921. Hu, X., Lee, E.-C., and Baker, N.E. (1995). Molecular analysis of scabrous mutant alleles from Drosophila melanogaster indicates a secreted protein with two functional domains. Genetics 141, 607–617. 1922. Huang, A.M. and Rubin, G.M. (2000). A misexpression screen identiﬁes genes that can modulate RAS1 pathway signaling in Drosophila melanogaster. Genetics 156, 1219– 1230. 1923. Huang, A.M., Rusch, J., and Levine, M. (1997). An anteroposterior Dorsal gradient in the Drosophila embryo. Genes Dev. 11, 1963–1973. 1924. Huang, F. (1998). Syntagms in development and evolution. Int. J. Dev. Biol. 42, 487–494. 1925. Huang, F., Dambly-Chaudi`ere, C., and Ghysen, A. (1991). The emergence of sense organs in the wing disc of Drosophila. Development 111, 1087–1095. 1926. Huang, F., van Helden, J., Dambly-Chaudi`ere, C., and Ghysen, A. (1995). Contribution of the gene extra-

359

1927.

1928.

1929.

1930.

1931.

1932.

1933.

1934.

1935.

1936.

1937.

1938.

1939.

1940.

1941.

1942.

1943.

macrochaetae to the precise positioning of bristles in Drosophila. Roux’s Arch. Dev. Biol. 204, 336–343. Huang, J.-D., Schwyter, D.H., Shirokawa, J.M., and Courey, A.J. (1993). The interplay between multiple enhancer and silencer elements deﬁnes the pattern of decapentaplegic expression. Genes Dev. 7, 694–704. Huang, M.-L., Hsu, C.-H., and Chien, C.-T. (2000). The proneural gene amos promotes multiple dendritic neuron formation in the Drosophila peripheral nervous system. Neuron 25, 57–67. Huang, X., Crute, B.E., Sun, C., Tang, Y.-Y., Kelley, J.J., III, Lewis, A.F., Hartman, K.L., Laue, T.M., Speck, N.A., and Bushweller, J.H. (1998). Overexpression, puriﬁcation, and biophysical characterization of the heterodimerization domain of the core-binding factor β subunit. J. Biol. Chem. 273 #4, 2480–2487. Huang, X., Peng, J.W., Speck, N.A., and Bushweller, J.H. (1999). Solution structure of core binding factor β and map of the CBFα binding site. Nature Struct. Biol. 6, 624–627. Huang, Y., Baker, R.T., and Fischer-Vize, J.A. (1995). Control of cell fate by a deubiquitinating enzyme encoded by the fat facets gene. Science 270, 1828–1831. Huang, Y. and Fischer-Vize, J.A. (1996). Undifferentiated cells in the developing Drosophila eye inﬂuence facet assembly and require the Fat facets ubiquitin-speciﬁc protease. Development 122, 3207–3216. Huang, Z. and Kunes, S. (1996). Hedgehog, transmitted along retinal axons, triggers neurogenesis in the developing visual centers of the Drosophila brain. Cell 86, 411–422. Huang, Z. and Kunes, S. (1998). Signals transmitted along retinal axons in Drosophila: Hedgehog signal reception and the cell circuitry of lamina cartridge assembly. Development 125, 3753–3764. Huang, Z.J., Edery, I., and Rosbash, M. (1993). PAS is a dimerization domain common to Drosophila Period and several transcription factors. Nature 364, 259–262. Huber, A.H., Nelson, W.J., and Weis, W.I. (1997). Threedimensional structure of the armadillo repeat region of β-catenin. Cell 90, 871–882. Huber, O., Bierkamp, C., and Kemler, R. (1996). Cadherins and catenins in development. Curr. Opin. Cell Biol. 8, 685– 691. Hudson, J.B., Podos, S.D., Keith, K., Simpson, S.L., and Ferguson, E.L. (1998). The Drosophila Medea gene is required downstream of dpp and encodes a functional homolog of human Smad4. Development 125, 1407–1420. Huggins, L.G., Cho, S.-H., Harrison, D., and Padgett, R.W. (2001). A dominant modiﬁer screen identiﬁes potential new components of the Dpp signal transduction pathway. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a162. Hughes, S.C. and Krause, H.M. (2001). Establishment and maintenance of parasegmental compartments. Development 128, 1109–1118. Huh, J.R., Yoo, S.J., and Hay, B.A. (2001). Visualizing activated caspases in Drosophila. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a315. Huiskes, R., Ruimerman, R., van Lenthe, G.H., and Janssen, J.D. (2000). Effects of mechanical forces on maintenance and adaptation of form in trabecular bone. Nature 405, 704–706. Hukriede, N.A. and Fleming, R.J. (1997). Beaded of Goldschmidt, an antimorphic allele of Serrate, encodes a protein lacking transmembrane and intracellular domains. Genetics 145, 359–374.

360

1944. Hukriede, N.A., Gu, Y., and Fleming, R.J. (1997). A dominant-negative form of Serrate acts as a general antagonist of Notch activation. Development 124, 3427–3437. ¨ 1945. Hulskamp, M., Schr¨oder, C., Pfeiﬂe, C., J¨ackle, H., and Tautz, D. (1989). Posterior segmentation of the Drosophila embryo in the absence of a maternal posterior organizer gene. Nature 338, 629–632. ¨ 1946. Hulskamp, M. and Tautz, D. (1991). Gap genes and gradients–the logic behind the gaps. BioEssays 13, 261– 268. ¨ 1947. Hulsken, J., Behrens, J., and Birchmeier, W. (1994). Tumorsuppressor gene products in cell contacts: the cadherinAPC-armadillo connection. Curr. Opin. Cell Biol. 6, 711– 716. 1948. Hummel, T., Krukkert, K., Roos, J., Davis, G., and Kl¨ambt, C. (2000). Drosophila Futsch/22C10 is a MAP1B-like protein required for dendritic and axonal development. Neuron 26, 357–370. 1949. Hunt, T. (1989). Cytoplasmic anchoring proteins and the control of nuclear localization. Cell 59, 949–951. 1950. Hunter, T. (2000). Signaling–2000 and beyond. Cell 100, 113–127. 1951. Huppert, S.S., Jacobsen, T.L., and Muskavitch, M.A.T. (1997). Feedback regulation is central to Delta-Notch signalling required for Drosophila wing vein morphogenesis. Development 124, 3283–3291. 1952. Hursh, D.A., Padgett, R.W., and Gelbart, W.M. (1993). Cross regulation of decapentaplegic and Ultrabithorax transcription in the embryonic visceral mesoderm of Drosophila. Development 117, 1211–1222. 1953. Huse, M., Chen, Y.-G., Massagu´e, J., and Kuriyan, J. (1999). Crystal structure of the cytoplasmic domain of the Type I TGFβ receptor in complex with FKBP12. Cell 96, 425–436. 1954. Huwe, A.W., Spillane, M.A., and Bryant, P.J. (2001). The nuts and bolts of the cell: PDZ-containing proteins and their potential binding partners. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a329. [See also 2001 Annu. Rev. Neurosci. 24, 1–29.] 1955. Huxley, J.S. and de Beer, G.R. (1934). “The Elements of Experimental Embryology.” Cambridge Univ. Pr., Cambridge. 1956. Hynes, R. (1985). Molecular biology of ﬁbronectin. Annu. Rev. Cell Biol. 1, 67–90. 1957. Hynes, R.O. (1999). Cell adhesion: old and new questions. Trends Genet. 15, M33–M37. 1958. Ichimura, T., Isobe, T., Okuyama, T., Takahashi, N., Araki, K., Kuwano, R., and Takahashi, Y. (1988). Molecular cloning of cDNA coding for brain-speciﬁc 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. Proc. Natl. Acad. Sci. USA 85, 7084– 7088. 1959. Ikeda, S., Kishida, S., Yamamoto, H., Murai, H., Koyama, S., and Kikuchi, A. (1998). Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3β and β-catenin and promotes GSK-3β-dependent phosphorylation of β-catenin. EMBO J. 17, 1371–1384. 1960. Incardona, J.P. and Eaton, S. (2000). Cholesterol in signal transduction. Curr. Opin. Cell Biol. 12, 193–203. 1961. Indrasamy, H., McKenzie, J.A., Woods, R., and Batterham, P. (2001). The contribution of Notch signalling pathway to bristle asymmetry. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a269. 1962. Ingham, P. (1988). The molecular genetics of embryonic pattern formation in Drosophila. Nature 335, 25–34.

REFERENCES

1963. Ingham, P., Martinez-Arias, A., Lawrence, P.A., and Howard, K. (1985). Expression of engrailed in the parasegment of Drosophila. Nature 317, 634–636. 1964. Ingham, P. and Smith, J. (1992). Crossing the threshold. Curr. Biol. 2, 465–467. 1965. Ingham, P.W. (1984). A gene that regulates the bithorax complex differentially in larval and adult cells of Drosophila. Cell 37, 815–823. 1966. Ingham, P.W. (1985). A clonal analysis of the requirement for the trithorax gene in the diversiﬁcation of segments in Drosophila. J. Embryol. Exp. Morph. 89, 349–365. 1967. Ingham, P.W. (1985). Genetic control of the spatial pattern of selector gene expression in Drosophila. Cold Spr. Harb. Symp. Quant. Biol. 50, 201–208. 1968. Ingham, P.W. (1991). Segment polarity genes and cell patterning within the Drosophila body segment. Curr. Op. Gen. Dev. 1, 261–267. 1969. Ingham, P.W. (1993). Localized hedgehog activity controls spatial limits of wingless transcription in the Drosophila embryo. Nature 366, 560–562. 1970. Ingham, P.W. (1994). Hedgehog points the way. Curr. Biol. 4, 347–350. 1971. Ingham, P.W. (1996). Has the quest for a Wnt receptor ﬁnally frizzled out? Trends Genet. 12, 382–384. 1972. Ingham, P.W. (1998). Boning up on Hedgehog’s movements. Nature 394, 16–17. 1973. Ingham, P.W. (1998). The patched gene in development and cancer. Curr. Opin. Gen. Dev. 8, 88–94. 1974. Ingham, P.W. (1998). Transducing Hedgehog: the story so far. EMBO J. 17, 3505–3511. [See also 2001 Genes Dev. 15, 3059–3087.] 1975. Ingham, P.W. (2000). Hedgehog signalling: how cholesterol modulates the signal. Curr. Biol. 10, R180–R183. 1976. Ingham, P.W. and Fietz, M.J. (1995). Quantitative effects of hedgehog and decapentaplegic activity on the patterning of the Drosophila wing. Curr. Biol. 5, 432–440. 1977. Ingham, P.W. and Hidalgo, A. (1993). Regulation of wingless transcription in the Drosophila embryo. Development 117, 283–291. 1978. Ingham, P.W. and Martinez Arias, A. (1992). Boundaries and ﬁelds in early embryos. Cell 68, 221–235. 1979. Ingham, P.W. and Nakano, Y. (1990). Cell patterning and segment polarity genes in Drosophila. Dev. Growth Differ. 32, 563–574. 1980. Ingham, P.W., Nystedt, S., Nakano, Y., Brown, W., Stark, D., van den Heuvel, M., and Taylor, A.M. (2000). Patched represses the Hedgehog signalling pathway by promoting modiﬁcation of the Smoothened protein. Curr. Biol. 10, 1315–1318. 1981. Ingham, P.W., Pinchin, S.M., Howard, K.R., and IshHorowicz, D. (1985). Genetic analysis of the hairy locus in Drosophila melanogaster. Genetics 111, 463–486. 1982. Ingham, P.W., Taylor, A.M., and Nakano, Y. (1991). Role of the Drosophila patched gene in positional signalling. Nature 353, 184–187. 1983. Inoue, H., Imamura, T., Ishidou, Y., Takase, M., Udagawa, Y., Oka, Y., Tsuneizumi, K., Tabata, T., Miyazono, K., and Kawabata, M. (1998). Interplay of signal mediators of Decapentaplegic (Dpp): molecular characterization of Mothers against dpp, Medea, and Daughters against dpp. Mol. Biol. Cell 9, 2145–2156. 1984. Ip, Y.T. and Hemavathy, K. (1997). Drosophila development: Delimiting patterns by repression. Curr. Biol. 7, R216–R218.

REFERENCES

1985. Ip, Y.T., Park, R.E., Kosman, D., Yazdanbakhsh, K., and Levine, M. (1992). dorsal-twist interactions establish snail expression in the presumptive mesoderm of the Drosophila embryo. Genes Dev. 6, 1518–1530. 1986. Irish, V., Lehmann, R., and Akam, M. (1989). The Drosophila posterior-group gene nanos functions by repressing hunchback activity. Nature 338, 646–648. 1987. Irish, V.F. and Gelbart, W.M. (1987). The decapentaplegic gene is required for dorsal-ventral patterning of the Drosophila embryo. Genes Dev. 1, 868–879. 1988. Irvine, K.D. (1999). Fringe, Notch, and making developmental boundaries. Curr. Opin. Gen. Dev. 9, 434–441. 1989. Irvine, K.D., Botas, J., Jha, S., Mann, R.S., and Hogness, D.S. (1993). Negative autoregulation by Ultrabithorax controls the level and pattern of its expression. Development 117, 387–399. 1990. Irvine, K.D., Helfand, S.L., and Hogness, D.S. (1991). The large upstream control region of the Drosophila homeotic gene Ultrabithorax. Development 111, 407–424. 1991. Irvine, K.D. and Vogt, T.F. (1997). Dorsal-ventral signaling in limb development. Curr. Opin. Cell Biol. 9, 867–876. 1992. Irvine, K.D. and Wieschaus, E. (1994). fringe, a boundaryspeciﬁc signaling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development. Cell 79, 595–606. 1993. Irwin, T. (1988). “Aristotle’s First Principles.” Clarendon Pr., Oxford. 1994. Isaksson, A., Peverali, F.A., Kockel, L., Mlodzik, M., and Bohmann, D. (1997). The deubiquitination enzyme Fat facets negatively regulates RTK/Ras/MAPK signalling during Drosophila eye development. Mechs. Dev. 68, 59–67. 1995. Ishimaru, S., Williams, R., Clark, E., Hanafusa, H., and Gaul, U. (1999). Activation of the Drosophila C3G leads to cell fate changes and overproliferation during development, mediated by the RAS-MAPK pathway and RAP1. EMBO J. 18, 145–155. 1996. Ishitani, T., Ninomiya-Tsuji, J., Nagai, S.-i., Nishita, M., Meneghini, M., Barker, N., Waterman, M., Bowerman, B., Clevers, H., Shibuya, H., and Matsumoto, K. (1999). The TAK1-NLK-MAPK-related pathway antagonizes signalling between β-catenin and transcription factor TCF. Nature 399, 798–802. 1997. Ives, P.T. (1935). The temperature-effective period of the scute-1 phenotype. Proc. Natl. Acad. Sci. USA 21, 646–650. 1998. Ives, P.T. (1939). The effects of high temperature on bristle frequencies in scute and wild-type males of Drosophila melanogaster. Genetics 24, 315–331. 1999. Iwanami, M. and Hiromi, Y. (2001). Regulating the neuronal induction by Argos and Sprouty in the eye development. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a256. 2000. Jack, J. and DeLotto, Y. (1995). Structure and regulation of a complex locus: the cut gene of Drosophila. Genetics 139, 1689–1700. 2001. Jack, J., Dorsett, D., Delotto, Y., and Liu, S. (1991). Expression of the cut locus in the Drosophila wing margin is required for cell type speciﬁcation and is regulated by a distant enhancer. Development 113, 735–747. 2002. Jack, T. and McGinnis, W. (1990). Establishment of the Deformed expression stripe requires the combinatorial action of coordinate, gap and pair-rule proteins. EMBO J. 9, 1187–1198. 2003. Jackson, P.D. and Hoffmann, F.M. (1994). Embryonic expression patterns of the Drosophila decapentaplegic gene:

361

2004.

2005. 2006.

2007.

2008.

2009. 2010.

2011.

2012.

2013.

2014.

2015.

2016.

2017. 2018.

2019.

2020.

separate regulatory elements control blastoderm expression and lateral ectodermal expression. Dev. Dynamics 199, 28–44. Jackson, S.M., Nakato, H., Sugiura, M., Jannuzi, A., Oakes, R., Kaluza, V., Golden, C., and Selleck, S.B. (1997). dally, a Drosophila glypican, controls cellular responses to the TGF-β-related morphogen, Dpp. Development 124, 4113– 4120. Jacob, F. (1977). Evolution and tinkering. Science 196, 1161–1166. Jacob, L., Opper, M., Metzroth, B., Phannavong, B., and Mechler, B.M. (1987). Structure of the l(2)gl gene of Drosophila and delimitation of its tumor suppressor domain. Cell 50, 215–225. Jacobs, D., Glossip, D., Xing, H., Muslin, A.J., and Kornfeld, K. (1999). Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase. Genes Dev. 13, 163–175. Jacobsen, T.L., Brennan, K., Martinez Arias, A., and Muskavitch, M.A.T. (1998). Cis-interactions between Delta and Notch modulate neurogenic signalling in Drosophila. Development 125, 4531–4540. [Cf. 2002 Dev. Biol. 241, 313–326.] Jacobson, M.D., Weil, M., and Raff, M.C. (1997). Programmed cell death in animal development. Cell 88, 347–354. Jacquemin, P., Hwang, J.-J., Martial, J.A., Doll´e, P., and Davidson, I. (1996). A novel family of developmentally regulated mammalian transcription factors containing the TEA/ATTS DNA binding domain. J. Biol. Chem. 271 #36, 21775–21785. Jaffe, L., Ryoo, H.-D., and Mann, R.S. (1997). A role for phosphorylation by casein kinase II in modulating Antennapedia activity in Drosophila. Genes Dev. 11, 1327–1340. Jagla, K., Bellard, M., and Frasch, M. (2001). A cluster of Drosophila homeobox genes involved in mesoderm differentiation programs. BioEssays 23, 125–133. Jagla, K., Jagla, T., Heitzler, P., Dretzen, G., Bellard, F., and Bellard, M. (1997). ladybird, a tandem of homeobox genes that maintain late wingless expression in terminal and dorsal epidermis of the Drosophila embryo. Development 124, 91–100. Jakobs, R., de Lorenzo, C., Spiess, E., Strand, D., and Mechler, B.M. (1996). Homo-oligomerization domains in the lethal(2)giant larvae tumor suppressor protein, p127 of Drosophila. J. Mol. Biol. 264, 484–496. James, A.A. and Bryant, P.J. (1981). Mutations causing pattern deﬁciencies and duplications in the imaginal wing disk of Drosophila melanogaster. Dev. Biol. 85, 39–54. James, R.M., Klerkx, A.H.E.M., Keighren, M., Flockhart, J.H., and West, J.D. (1995). Restricted distribution of tetraploid cells in mouse tetraploid-diploid chimaeras. Dev. Biol. 167, 213–226. Jan, Y.-N. and Jan, L.Y. (2000). Polarity in cell division: what frames thy fearful symmetry? Cell 100, 599–602. Jan, Y.N. and Jan, L.Y. (1993). The peripheral nervous system. In “The Development of Drosophila melanogaster,” Vol. 2 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Lab. Pr.: Plainview, N. Y., pp. 1207–1244. Jan, Y.N. and Jan, L.Y. (1994). Genetic control of cell fate speciﬁcation in Drosophila peripheral nervous system. Annu. Rev. Genet. 28, 373–393. Jan, Y.N. and Jan, L.Y. (1995). Maggot’s hair and bug’s eye: role of cell interactions and intrinsic factors in cell fate speciﬁcation. Neuron 14, 1–5.

362

2021. Jan, Y.N. and Jan, L.Y. (1998). Asymmetric cell division. Nature 392, 775–778. 2022. Jang, C.C. and Sun, Y.H. (2001). Molecular analysis of the eyg-toe gene complex in Drosophila. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a198. 2023. Janknecht, R. and Hunter, T. (1996). A growing coactivator network. Nature 383, 22–23. 2024. Janknecht, R. and Hunter, T. (1996). Transcriptional control: versatile molecular glue. Curr. Biol. 6, 951–954. 2025. Janknecht, R., Sander, C., and Pongs, O. (1991). (HX)n repeats: a pH-controlled protein-protein interaction motif of eukaryotic transcription factors? FEBS Letters 295, 1–2. 2026. Janning, W. (1978). Gynandromorph fate maps in Drosophila. In “Genetic Mosaics and Cell Differentiation,” Results and Problems in Cell Differentiation, Vol. 9, (W.J. Gehring, ed.). Springer-Verlag: Berlin, pp. 1–28. 2027. Janning, W. (1997). FlyView, a Drosophila image database, and other Drosophila databases. Sems. Cell Dev. Biol. 8, 469–475. 2028. Janning, W., Labhart, C., and N¨othiger, R. (1983). Cell lineage restrictions in the genital disc of Drosophila revealed by Minute gynandromorphs. Roux’s Arch. Dev. Biol. 192, 337–346. 2029. Janning, W., Pfreundt, J., and Tiemann, R. (1979). The distribution of anlagen in the early embryo of Drosophila. In “Cell Lineage, Stem Cells and Cell Determination,” (N. Le Douarin, ed.). North-Holland Pub. Co.: Amsterdam, pp. 83–98. 2030. Jans, D.A. (1994). Nuclear signaling pathways for polypeptide ligands and their membrane receptors? FASEB J. 8, 841–847. [See also 2001 Trends Genet. 17, 625–626.] 2031. Jans, D.A. and Hassan, G. (1998). Nuclear targeting by growth factors, cytokines, and their receptors: a role in signaling? BioEssays 20, 400–411. [See also 2001 Cell 106, 1–4.] ¨ 2032. Jans, D.A. and Hubner, S. (1996). Regulation of protein transport to the nucleus: central role of phosphorylation. Physiol. Rev. 76, 651–685. 2033. Jans, D.A., Xiao, C.-Y., and Lam, M.H.C. (2000). Nuclear targeting signal recognition: a key control point in nuclear transport? BioEssays 22, 532–544. 2034. Janssens, H. and Gehring, W.J. (1999). Isolation and characterization of drosocrystallin, a lens crystallin gene of Drosophila melanogaster. Dev. Biol. 207, 204–214. 2035. Jantzen, H.-M., Admon, A., Bell, S.P., and Tjian, R. (1990). Nucleolar transcription factor hUBF contains a DNAbinding motif with homology to HMG proteins. Nature 344, 830–836. 2036. Jarman, A.P. (1996). Epithelial polarity in the Drosophila compound eye: eyes left or right? Trends Genet. 12, 121– 123. 2037. Jarman, A.P. (2000). Developmental genetics: Vertebrates and insects see eye to eye. Curr. Biol. 10, R857–R859. 2038. Jarman, A.P. and Ahmed, I. (1998). The speciﬁcity of proneural genes in determining Drosophila sense organ identity. Mechs. Dev. 76, 117–125. 2039. Jarman, A.P., Brand, M., Jan, L.Y., and Jan, Y.N. (1993). The regulation and function of the helix-loop-helix gene, asense, in Drosophila neural precursors. Development 119, 19–29. 2040. Jarman, A.P., Grau, Y., Jan, L.Y., and Jan, Y.N. (1993). atonal is a proneural gene that directs chordotonal organ formation in the Drosophila peripheral nervous system. Cell 73, 1307–1321.

REFERENCES

2041. Jarman, A.P., Grell, E.H., Ackerman, L., Jan, L.Y., and Jan, Y.N. (1994). atonal is the proneural gene for Drosophila photoreceptors. Nature 369, 398–400. 2042. Jarman, A.P., Sun, Y., Jan, L.Y., and Jan, Y.N. (1995). Role of the proneural gene, atonal, in formation of Drosophila chordotonal organs and photoreceptors. Development 121, 2019–2030. 2043. Jarriault, S., Brou, C., Logeat, F., Schroeter, E.H., Kopan, R., and Israel, A. (1995). Signalling downstream of activated mammalian Notch. Nature 377, 355–358. 2044. J¨arvilehto, M. (1985). The eye: vision and perception. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology,” Vol. 6 (G.A. Kerkut and L.I. Gilbert, eds.). Pergamon Pr.: New York, pp. 355–429. 2045. Jaw, T.J., You, L.-R., Knoepﬂer, P.S., Yao, L.-C., Pai, C.-Y., Tang, C.-Y., Chang, L.-P., Berthelsen, J., Blasi, F., Kamps, M.P., and Sun, Y.H. (2000). Direct interaction of two homeoproteins, Homothorax and Extradenticle, is essential for EXD nuclear localization and function. Mechs. Dev. 91, 279–291. 2046. Jayaraman, P.-S., Hirst, K., and Goding, C.R. (1994). The activation domain of a basic helix-loop-helix protein is masked by repressor interaction with domains distinct from that required for transcription regulation. EMBO J. 13, 2192–2199. 2047. Jaynes, J.B. and O’Farrell, P.H. (1988). Activation and repression of transcription by homoeodomain-containing proteins that bind a common site. Nature 336, 744–749. 2048. Jaynes, J.B. and O’Farrell, P.H. (1991). Active repression of transcription by the Engrailed homeodomain protein. EMBO J. 10, 1427–1433. 2049. Jazwinska, A., Kirov, N., Wieschaus, E., Roth, S., and Rushlow, C. (1999). The Drosophila gene brinker reveals a novel mechanism of Dpp target gene regulation. Cell 96, 563–573. 2050. Jazwinska, A., Rushlow, C., and Roth, S. (1999). The role of brinker in mediating the graded response to Dpp in early Drosophila embryos. Development 126, 3323–3334. 2051. Jen, W.-C., Gawantka, V., Pollet, N., Niehrs, C., and Kintner, C. (1999). Periodic repression of Notch pathway genes governs the segmentation of Xenopus embryos. Genes Dev. 13, 1486–1499. 2052. Jennings, B., de Celis, J., Delidakis, C., Preiss, A., and Bray, S. (1995). Role of Notch and achaete-scute complex in the expression of Enhancer of split bHLH proteins. Development 121, 3745–3752. 2053. Jennings, B., Preiss, A., Delidakis, C., and Bray, S. (1994). The Notch signalling pathway is required for Enhancer of split bHLH protein expression during neurogenesis in the Drosophila embryo. Development 120, 3537–3548. 2054. Jennings, B.H., Tyler, D.M., and Bray, S.J. (1999). Target speciﬁcities of Drosophila Enhancer of split basic helixloop-helix proteins. Mol. Cell. Biol. 19, 4600–4610. 2055. Jhaveri, D., Sen, A., Reddy, V., and Rodrigues, V. (2000). Sense organ identity in the Drosophila antenna is speciﬁed by the expression of the proneural gene atonal. Mechs. Dev. 99, 101–111. 2056. Jhaveri, D., Sen, A., and Rodrigues, V. (2000). Mechanisms underlying olfactory neuronal connectivity in Drosophila – the Atonal lineage organizes the periphery while sensory neurons and glia pattern the olfactory lobe. Dev. Biol. 226, 73–87. 2057. Jiang, J., Hoey, T., and Levine, M. (1991). Autoregulation of a segmentation gene in Drosophila: combinatorial

REFERENCES

2058.

2059.

2060.

2061.

2062. 2063.

2064.

2065.

2066.

2067.

2068.

2069.

2070.

2071.

2072.

2073.

interaction of the even-skipped homeo box protein with a distal enhancer element. Genes Dev. 5, 265–277. Jiang, J. and Struhl, G. (1995). Protein kinase A and Hedgehog signaling in Drosophila limb development. Cell 80, 563–572. Jiang, J. and Struhl, G. (1996). Complementary and mutually exclusive activities of Decapentaplegic and Wingless organize axial patterning during Drosophila leg development. Cell 86, 401–409. Jiang, J. and Struhl, G. (1998). Regulation of the Hedgehog and Wingless signalling pathways by the F-box/WD40repeat protein Slimb. Nature 391, 493–496. Jiang, Y.-J., Aerne, B.L., Smithers, L., Haddon, C., IshHorowicz, D., and Lewis, J. (2000). Notch signalling and the synchronization of the somite segmentation clock. Nature 408, 475–479. Jim´enez, F. and Modolell, J. (1993). Neural fate speciﬁcation in Drosophila. Curr. Op. Gen. Dev. 3, 626–632. Jim´enez, G. and Ish-Horowicz, D. (1997). A chimeric Enhancer-of-split transcriptional activator drives neural development and achaete-scute expression. Mol. Cell. Biol. 17, 4355–4362. Jim´enez, G., Paroush, Z., and Ish-Horowicz, D. (1997). Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed. Genes Dev. 11, 3072–3082. Jim´enez, G., Pinchin, S.M., and Ish-Horowicz, D. (1996). In vivo interactions of the Drosophila Hairy and Runt transcriptional repressors with target promoters. EMBO J. 15, 7088–7098. Jim´enez, G., Verrijzer, C.P., and Ish-Horowicz, D. (1999). A conserved motif in Goosecoid mediates Grouchodependent repression in Drosophila embryos. Mol. Cell. Biol. 19, 2080–2087. Jin, M.-H., Sawamoto, K., Ito, M., and Okano, H. (2000). The interaction between the Drosophila secreted protein Argos and the Epidermal Growth Factor Receptor inhibits dimerization of the receptor and binding of secreted Spitz to the receptor. Mol. Cell. Biol. 20, 2098–2107. Joazeiro, C.A.P., Wing, S.S., Huang, H.-k., Leverson, J.D., Hunter, T., and Liu, Y.-C. (1999). The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science 286, 309–312. Jockusch, E.L., Nulsen, C., Newfeld, S.J., and Nagy, L.M. (2000). Leg development in ﬂies versus grasshoppers: differences in dpp expression do not lead to differences in the expression of downstream components of the leg patterning pathway. Development 127, 1617– 1626. Johansen, K.M., Fehon, R.G., and Artavanis-Tsakonas, S. (1989). The Notch gene product is a glycoprotein expressed on the cell surface of both epidermal and neuronal precursor cells during Drosophila development. J. Cell Biol. 109, 2427–2440. John, A., Smith, S.T., and Jaynes, J.B. (1995). Inserting the Ftz homeodomain into Engrailed creates a dominant transcriptional repressor that speciﬁcally turns off Ftz target genes in vivo. Development 121, 1801–1813. Johnson, R.L., Grenier, J.K., and Scott, M.P. (1995). patched overexpression alters wing disc size and pattern: transcriptional and post-transcriptional effects on hedgehog targets. Development 121, 4161–4170. Johnson, R.L., Milenkovic, L., and Scott, M.P. (2000). In vivo functions of the Patched protein: requirement of the

363

2074.

2075.

2076. 2077.

2078. 2079.

2080.

2081.

2082.

2083. 2084.

2085.

2086.

2087.

2088.

2089.

2090.

2091. 2092.

C terminus for target gene inactivation but not Hedgehog sequestration. Molec. Cell 6, 467–478. Johnson, R.L. and Scott, M.P. (1997). Control of cell growth and fate by patched genes. Cold Spr. Harb. Symp. Quant. Biol. 62, 205–215. Johnson, R.L. and Scott, M.P. (1998). New players and puzzles in the Hedgehog signaling pathway. Curr. Opin. Gen. Dev. 8, 450–456. Johnson, R.L. and Tabin, C. (1995). The long and short of hedgehog signaling. Cell 81, 313–316. Johnson, S.A. and Milner, M.J. (1987). The ﬁnal stages of wing development in Drosophila melanogaster. Tissue Cell 19, 505–513. Johnston, L.A. (1998). Uncoupling growth from the cell cycle. BioEssays 20, 283–286. Johnston, L.A. and Edgar, B.A. (1998). Wingless and Notch regulate cell-cycle arrest in the developing Drosophila wing. Nature 394, 82–84. Johnston, L.A., Ostrow, B.D., Jasoni, C., and Blochlinger, K. (1998). The homeobox gene cut interacts genetically with the homeotic genes proboscipedia and Antennapedia. Genetics 149, 131–142. Johnston, L.A., Prober, D.A., Edgar, B.A., Eisenman, R.N., and Gallant, P. (1999). Drosophila myc regulates cellular growth during development. Cell 98, 779–790. Johnston, L.A. and Schubiger, G. (1996). Ectopic expression of wingless in imaginal discs interferes with decapentaplegic expression and alters cell determination. Development 122, 3519–3529. Johnston, N.V. (1966). The pattern of abdominal microchaetae in Drosophila. Aust. J. Biol. Sci. 19, 155–166. Johnston, S.H., Rauskolb, C., Wilson, R., Prabhakaran, B., Irvine, K.D., and Vogt, T.F. (1997). A family of mammalian Fringe genes implicated in boundary determination and the Notch pathway. Development 124, 2245–2254. Joliot, A., Maizel, A., Rosenberg, D., Trembleau, A., Dupas, S., Volovitch, M., and Prochiantz, A. (1998). Identiﬁcation of a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein. Curr. Biol. 8, 856– 863. Jones, C., Reifegerste, R., and Moses, K. (2001). Molecular and functional characterization of a dominant modiﬁer of hedgehog. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a199. Jones, D.H., Ley, S., and Aitken, A. (1995). Isoforms of 14-3-3 protein can form homo- and heterodimers in vivo and in vitro: implications for function as adapter proteins. FEBS Letters 368, 55–58. Jones, K.H., Liu, J., and Adler, P.N. (1996). Molecular analysis of EMS-induced frizzled mutations in Drosophila melanogaster. Genetics 142, 205–215. Jones, N. (1990). Transcriptional regulation by dimerization: two sides to an incestuous relationship. Cell 61, 9–11. Jones, N.A., Kuo, Y.M., Sun, Y.H., and Beckendorf, S.K. (1998). The Drosophila Pax gene eye gone is required for embryonic salivary gland duct development. Development 125, 4163–4174. Jones, S. and Thornton, J.M. (1996). Principles of proteinprotein interactions. Proc. Natl. Acad. Sci. USA 93, 13–20. J¨onsson, F. and Knust, E. (1996). Distinct functions of the Drosophila genes Serrate and Delta revealed by ectopic expression during wing development. Dev. Genes Evol. 206, 91–101.

364

2093. Jordan, J.D., Landau, E.M., and Iyengar, R. (2000). Signaling networks: the origins of cellular multitasking. Cell 103, 193–200. 2094. Jorgensen, E.M. and Garber, R.L. (1987). Function and misfunction of the two promoters of the Drosophila Antennapedia gene. Genes Dev. 1, 544–555. 2095. Jouve, C., Palmeirim, I., Henrique, D., Beckers, J., Gossler, A., Ish-Horowicz, D., and Pourqui´e, O. (2000). Notch signalling is required for cyclic expression of the hairy-like gene HES1 in the presomitic mesoderm. Development 127, 1421–1429. [See also 2001 Genes Dev. 15, 2642–2647.] 2096. Ju, B.-G., Jeong, S., Bae, E., Hyun, S., Carroll, S.B., Yim, J., and Kim, J. (2000). Fringe forms a complex with Notch. Nature 405, 191–195. 2097. Judd, B.H. (1998). Genes and chromomeres: a puzzle in three dimensions. Genetics 150, 1–9. 2098. Jun, S. and Desplan, C. (1996). Cooperative interactions between paired domain and homeodomain. Development 122, 2639–2650. 2099. Jun, S., Wallen, R.V., Goriely, A., Kalionis, B., and Desplan, C. (1998). Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition. Proc. Natl. Acad. Sci. USA 95, 13720–13725. 2100. Jung, H.-S., Francis-West, P.H., Widelitz, R.B., Jiang, T.-X., Ting-Berreth, S., Tickle, C., Wolpert, L., and Chuong, C.-M. (1998). Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning. Dev. Biol. 196, 11–23. ¨ 2101. Jurgens, G. (1988). Head and tail development of the Drosophila embryo involves spalt, a novel homeotic gene. EMBO J. 7, 189–196. ¨ 2102. Jurgens, G. and Gateff, E. (1979). Pattern speciﬁcation in imaginal discs of Drosophila melanogaster. Developmental analysis of a temperature-sensitive mutant producing duplicated legs. W. Roux’s Arch. 186, 1–25. ¨ 2103. Jurgens, G. and Hartenstein, V. (1993). The terminal regions of the body pattern. In “The Development of Drosophila melanogaster,” Vol. 1 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Lab. Pr.: Plainview, N. Y., pp. 687–746. ¨ ¨ 2104. Jurgens, G., Lehmann, R., Schardin, M., and NussleinVolhard, C. (1986). Segmental organisation of the head in the embryo of Drosophila melanogaster. Roux’s Arch. Dev. Biol. 195, 359–377. ¨ ¨ 2105. Jurgens, G., Wieschaus, E., Nusslein-Volhard, C., and Kluding, H. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. II. Zygotic loci on the third chromosome. Roux’s Arch. Dev. Biol. 193, 283– 295. 2106. Jursnich, V.A. and Burtis, K.C. (1993). A positive role in differentiation for the male doublesex protein of Drosophila. Dev. Biol. 155, 235–249. 2107. Jursnich, V.A., Fraser, S.E., Held, L.I., Jr., Ryerse, J., and Bryant, P.J. (1990). Defective gap-junctional communication associated with imaginal disc overgrowth and degeneration caused by mutations of the dco gene in Drosophila. Dev. Biol. 140, 413–429. ¨ 2108. Kadlec, L., Ghabrial, A., and Schupbach, T. (2001). The gritz gene encodes a novel putative ligand of the Drosophila Egf receptor. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a152. 2109. Kadonaga, J.T. (1998). Eukaryotic transcription: an interlaced network of transcription factors and chromatinmodifying machines. Cell 92, 307–313.

REFERENCES

2110. Kadowaki, T., Wilder, E., Klingensmith, J., Zachary, K., and Perrimon, N. (1996). The segment polarity gene porcupine encodes a putative multitransmembrane protein involved in Wingless processing. Genes Dev. 10, 3116–3128. 2111. Kafatos, F.C. (1976). Sequential cell polymorphism: a fundamental concept in developmental biology. Adv. Insect Physiol. 12, 1–15. 2112. Kagoshima, H., Akamatsu, Y., Ito, Y., and Shigesada, K. (1996). Functional dissection of the α and β subunits of transcription factor PEBP2 and the redox susceptibility of its DNA binding activity. J. Biol. Chem. 271 #51, 33074– 33082. 2113. Kagoshima, H., Shigesada, K., Satake, M., Ito, Y., Miyoshi, H., Ohki, M., Pepling, M., and Gergen, P. (1993). The Runt domain identiﬁes a new family of heteromeric transcriptional regulators. Trends Genet. 9, 338–341. 2114. Kal, A.J., Mahmoudi, T., Zak, N.B., and Verrijzer, C.P. (2000). The Drosophila Brahma complex is an essential coactivator for the trithorax group protein Zeste. Genes Dev. 14, 1058–1071. 2115. Kalderon, D. (1995). Responses to Hedgehog. Curr. Biol. 5, 580–582. 2116. Kalderon, D. (1997). Hedgehog signalling: Ci complex cuts and clasps. Curr. Biol. 7, R759–R762. 2117. Kalderon, D. (2000). Transducing the Hedgehog signal. Cell 103, 371–374. 2118. Kalderon, D. and Rubin, G.M. (1988). Isolation and characterization of Drosophila cAMP-dependent protein kinase genes. Genes Dev. 2, 1539–1556. 2119. Kalionis, B. and O’Farrell, P.H. (1993). A universal target sequence is bound in vitro by diverse homeodomains. Mechs. Dev. 43, 57–70. 2120. Kalmes, A., Merdes, G., Neumann, B., Strand, D., and Mechler, B.M. (1996). A serine-kinase associated with the p127-l(2)gl tumour suppressor of Drosophila may regulate the binding of p127 to nonmuscle myosin II heavy chain and the attachment of p127 to the plasma membrane. J. Cell Sci. 109, 1359–1368. 2121. Kalthoff, K. (2001). “Analysis of Biological Development.” McGraw-Hill, New York. 2122. Kamachi, Y., Uchikawa, M., and Kondoh, H. (2000). Pairing SOX off with partners in the regulation of embryonic development. Trends Genet. 16, 182–187. 2123. Kamashev, D.E., Balandina, A.V., and Karpov, V.L. (2000). Tramtrack protein-DNA interactions. J. Biol. Chem. 275 #46, 36056–36061. 2124. Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S.-C., Heyman, R.A., Rose, D.W., Glass, C.K., and Rosenfeld, M.G. (1996). A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85, 403–414. 2125. Kammermeyer, K.L. and Wadsworth, S.C. (1987). Expression of Drosophila epidermal growth factor receptor homologue in mitotic cell populations. Development 100, 201–210. 2126. Kango-Singh, M., Singh, A., and Sun, Y.H. (2001). Requirement of hedgehog (hh) for the induction of ectopic eyes by the Drosophila PAX6 gene–eyeless (ey). Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a47. 2127. Kania, M.A., Bonner, A.S., Duffy, J.B., and Gergen, J.P. (1990). The Drosophila segmentation gene runt encodes a novel nuclear regulatory protein that is also expressed in the developing nervous system. Genes Dev. 4, 1701–1713. ´ A., Garen, S.H., Harte, P.J., and Lewis, 2128. Kankel, D.R., Ferrus,

REFERENCES

2129.

2130.

2131.

2132.

2133.

2134.

2135.

2136.

2137.

2138. 2139.

2140.

2141.

2142.

2143.

2144.

2145.

P.E. (1980). The structure and development of the nervous system. In “The Genetics and Biology of Drosophila,” Vol. 2d (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 295–368. Kao, H.-Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner, C.R., Evans, R.M., and Kadesch, T. (1998). A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev. 12, 2269–2277. [See also 2001 Curr. Biol. 11, 789-792.] Kaphingst, K. and Kunes, S. (1994). Pattern formation in the visual centers of the Drosophila brain: wingless acts via decapentaplegic to specify the dorsoventral axis. Cell 78, 437–448. Karim, F.D., Chang, H.C., Therrien, M., Wassarman, D.A., Laverty, T., and Rubin, G.M. (1996). A screen for genes that function downstream of Ras1 during Drosophila eye development. Genetics 143, 315–329. Karim, F.D. and Rubin, G.M. (1998). Ectopic expression of activated Ras1 induces hyperplastic growth and increased cell death in Drosophila imaginal tissues. Development 125, 1–9. Karim, F.D. and Rubin, G.M. (1999). PTP-ER, a novel tyrosine phosphatase, functions downstream of Ras1 to downregulate MAP kinase during Drosophila eye development. Molec. Cell 3, 741–750. Karim, F.D. and Thummel, C.S. (1991). Ecdysone coordinates the timing and amounts of E74A and E74B transcription in Drosophila. Genes Dev. 5, 1067–1079. Karim, F.D., Urness, L.D., Thummel, C.S., Klemsz, M.J., McKercher, S.R., Celada, A., Van Beveren, C., Maki, R.A., Gunther, C.V., Nye, J.A., and Graves, B.J. (1990). The ETSdomain: a new DNA-binding motif that recognizes a purine-rich core DNA sequence. Genes Dev. 4, 1451–1453. Karin, M. and Hunter, T. (1995). Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr. Biol. 5, 747–757. Karlovich, C.A., Bonﬁni, L., McCollam, L., Rogge, R.D., Daga, A., Czech, M.P., and Banerjee, U. (1995). In vivo functional analysis of the Ras exchange factor Son of sevenless. Science 268, 576–579. Karlsson, J. (1979). A major difference between transdetermination and homeosis. Nature 279, 426–428. Karlsson, J. (1980). Distal regeneration in proximal fragments of the wing disc of Drosophila. J. Embryol. Exp. Morph. 59, 315–323. Karlsson, J. (1981). The distribution of regenerative potential in the wing disc of Drosophila. J. Embryol. Exp. Morph. 61, 303–316. Karlsson, J. (1981). Sequence of regeneration in the Drosophila wing disc. J. Embryol. Exp. Morph. 65 (Suppl.), 37–47. Karlsson, J. (1984). Morphogenesis and compartments in Drosophila. In “Pattern Formation: A Primer in Developmental Biology,” (G.M. Malacinski and S.V. Bryant, eds.). Macmillan: New York, pp. 323–337. Karlsson, J. and Smith, R.J. (1981). Regeneration from duplicating fragments of the Drosophila wing disc. J. Embryol. Exp. Morph. 66, 117–126. Karpen, G.H. and Schubiger, G. (1981). Extensive regulatory capabilities of a Drosophila imaginal disk blastema. Nature 294, 744–747. Karpilow, J., Kolodkin, A., Bork, T., and Venkatesh, T. (1989). Neuronal development in the Drosophila compound eye: rap gene function is required in photorecep-

365

2146.

2147.

2148.

2149.

2150.

2151.

2152.

2153.

2154. 2155.

2156. 2157.

2158.

2159. 2160.

2161.

2162. 2163.

tor cell R8 for ommatidial pattern formation. Genes Dev. 3, 1834–1844. Karr, T.L., Weir, M.P., Ali, Z., and Kornberg, T. (1989). Patterns of engrailed protein in early Drosophila embryos. Development 105, 605–612. Katsani, K.R., Hajibagheri, M.A.N., and Verrijzer, C.P. (1999). Co-operative DNA binding by GAGA transcription factor requires the conserved BTB/POZ domain and reorganizes promoter topology. EMBO J. 18, 698–708. Katsube, T., Takahisa, M., Ueda, R., Hashimoto, N., Kobayashi, M., and Togashi, S. (1998). Cortactin associates with the cell-cell junction protein ZO-1 in both Drosophila and mouse. J. Biol. Chem. 273 #45, 29672–29677. Katz, M.J. and Grenander, U. (1982). Developmental matching and the numerical matching hypothesis for neuronal cell death. J. Theor. Biol. 98, 501–517. Katz, M.J. and Lasek, R.J. (1978). Evolution of the nervous system: role of ontogenetic mechanisms in the evolution of matching populations. Proc. Natl. Acad. Sci. USA 75, 1349–1352. Katzen, A.L., Jackson, J., Harmon, B.P., Fung, S.-M., Ramsay, G., and Bishop, J.M. (1998). Drosophila myb is required for the G2 /M transition and maintenance of diploidy. Genes Dev. 12, 831–843. Katzen, A.L., Kornberg, T., and Bishop, J.M. (1991). Expression during Drosophila development of DER, a gene related to erbB-1 and neu: correlations with mutant phenotypes. Dev. Biol. 145, 287–301. Kauffman, S. (1975). Control circuits for determination and transdetermination: interpreting positional information in a binary epigenetic code. In “Cell Patterning,” Ciba Found. Symp., Vol. 29 (R. Porter and J. Rivers, eds.). Elsevier: Amsterdam, pp. 201–221. Kauffman, S. (1996). Evolving evolvability. Nature 382, 309–310. Kauffman, S.A. (1971). Gene regulation networks: a theory for their global structure and behaviors. Curr. Top. Dev. Biol. 6, 145–182. Kauffman, S.A. (1973). Control circuits for determination and transdetermination. Science 181, 310–318. Kauffman, S.A. (1981). Bifurcations in insect morphogenesis. I. In “Nonlinear Phenomena in Physics and Biology,” (R.H. Enns, B.L. Jones, R.M. Miura, and S.S. Rangnekar, eds.). Plenum: New York, pp. 401–450. Kauffman, S.A. (1981). Bifurcations in insect morphogenesis. II. In “Nonlinear Phenomena in Physics and Biology,” (R.H. Enns, B.L. Jones, R.M. Miura, and S.S. Rangnekar, eds.). Plenum: New York, pp. 451–484. Kauffman, S.A. (1981). Pattern formation in the Drosophila embryo. Phil. Trans. Roy. Soc. Lond. B 295, 567–594. Kauffman, S.A. (1983). Developmental constraints: internal factors in evolution. In “Development and Evolution,” Symp. Brit. Soc. Dev. Biol., Vol. 6 (B.C. Goodwin, N. Holder, and C.C. Wylie, eds.). Cambridge Univ. Pr.: Cambridge, pp. 195–225. Kauffman, S.A. (1984). Pattern generation and regeneration. In “Pattern Formation: A Primer in Developmental Biology,” (G.M. Malacinski and S.V. Bryant, eds.). Macmillan: New York, pp. 73–102. Kauffman, S.A. (1987). Developmental logic and its evolution. BioEssays 6, 82–87. Kauffman, S.A. (1993). “The Origins of Order: Selforganization and Selection in Evolution.” Oxford Univ. Pr., Oxford.

366

2164. Kauffman, S.A., Shymko, R.M., and Trabert, K. (1978). Control of sequential compartment formation in Drosophila. Science 199, 259–270. 2165. Kauffmann, R.C., Li, S., Gallagher, P.A., Zhang, J., and Carthew, R.W. (1996). Ras1 signaling and transcriptional competence in the R7 cell of Drosophila. Genes Dev. 10, 2167–2178. 2166. Kaufman, T.C. (1983). The genetic regulation of segmentation in Drosophila melanogaster. In “Time, Space, and Pattern in Embryonic Development,” (W.R. Jeffery and R.A. Raff, eds.). Alan R. Liss: New York, pp. 365–383. 2167. Kaufman, T.C., Seeger, M.A., and Olsen, G. (1990). Molecular and genetic organization of the Antennapedia gene complex of Drosophila melanogaster. Adv. Genet. 27, 309– 362. 2168. Kavaler, J., Fu, W., Duan, H., Noll, M., and Posakony, J.W. (1999). An essential role for the Drosophila Pax2 homolog in the differentiation of adult sensory organs. Development 126, 2261–2272. 2169. Kazemi-Esfarjani, P. and Benzer, S. (2000). Genetic suppression of polyglutamine toxicity in Drosophila. Science 287, 1837–1840. 2170. Kehl, B.T., Cho, K.-O., and Choi, K.-W. (1998). mirror, a Drosophila homeobox gene in the iroquois complex, is required for sensory organ and alula formation. Development 125, 1217–1227. 2171. Kehle, J., Beuchle, D., Treuheit, S., Christen, B., Kennison, ¨ J.A., Bienz, M., and Muller, J. (1998). dMi-2, a Hunchbackinteracting protein that functions in Polycomb repression. Science 282, 1897–1900. 2172. Keightley, P.D. (1995). Loci with large effects. Curr. Biol. 5, 485–487. 2173. Keil, T.A. (1997). Functional morphology of insect mechanoreceptors. Microsc. Res. Tech. 39, 506–531. 2174. Keil, T.A. and Steinbrecht, R.A. (1984). Mechanosensitive and olfactory sensilla of insects. In “Insect Ultrastructure,” Vol. 2 (R.C. King and H. Akai, eds.). Plenum: New York, pp. 477–516. 2175. Keil, T.A. and Steiner, C. (1990). Morphogenesis of the antenna of the male silkmoth, Antheraea polyphemus. II. Differential mitoses of “dark” precursor cells create the anlagen of sensilla. Tissue & Cell 22, 705–720. 2176. Keisman, E.L. and Baker, B.S. (2001). The Drosophila sex determination hierarchy modulates wingless and decapentaplegic signaling to deploy dachshund sexspeciﬁcally in the genital imaginal disc. Development 128, 1643–1656. 2177. Keller, C.A., Erickson, M.S., and Abmayr, S.M. (1997). Misexpression of nautilus induces myogenesis in cardioblasts and alters the pattern of somatic muscle ﬁbers. Dev. Biol. 181, 197–212. 2178. Keller, E.F. (1996). Drosophila embryos as transitional objects: the work of Donald Poulson and Christiane ¨ Nusslein-Volhard. Hist. Studies Phys. Biol. Sci. 26, 313– 346. 2179. Keller, E.F. (2000). Decoding the genetic program ... or, some circular logic in the logic of circularity. In “The Concept of the Gene in Development and Evolution: Historical and Epistemological Perspectives,” (P.J. Beurton, R. Falk, and H.-J. Rheinberger, eds.). Cambridge Univ. Pr.: Cambridge, pp. 159–177. 2180. Keller, S.A., Mao, Y., Strufﬁ, P., Margulies, C., Yurk, C.E., Anderson, A.R., Amey, R.L., Moore, S., Ebels, J.M., Foley, K., Corado, M., and Arnosti, D.N. (2000). dCtBP-dependent

REFERENCES

2181.

2182.

2183.

2184. 2185.

2186.

2187.

2188.

2189.

2190.

2191. 2192. 2193.

2194.

2195.

2196.

2197. 2198.

2199.

2200.

and -independent repression activities of the Drosophila Knirps protein. Mol. Cell. Biol. 20, 7247–7258. Kellerman, K.A., Mattson, D.M., and Duncan, I. (1990). Mutations affecting the stability of the fushi tarazu protein of Drosophila. Genes Dev. 4, 1936–1950. Kelley, M.R., Kidd, S., Deutsch, W.A., and Young, M.W. (1987). Mutations altering the structure of epidermal growth factor-like coding sequences at the Drosophila Notch locus. Cell 51, 539–548. Kelley, R.L. and Kuroda, M.I. (2001). The MSL dosage compensation complex modiﬁes chromatin when redirected to autosomes. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a102. [Cf. 2001 EMBO J. 20, 2236–2245.] Kelly, R.B. (1995). Ringing necks with dynamin. Nature 374, 116–117. Kemler, R. (1993). From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. Trends Genet. 9, 317–321. Kenan, D.J., Query, C.C., and Keene, J.D. (1991). RNA recognition: towards identifying determinants of speciﬁcity. Trends Biochem. Sci. 16, 214–220. Kennerdell, J.R. and Carthew, R.W. (1998). Use of dsRNAmediated genetic interference to demonstrate that frizzled and frizzled 2 act in the Wingless pathway. Cell 95, 1017–1026. Kennison, J.A. (1993). Transcriptional activation of Drosophila homeotic genes from distant regulatory elements. Trends Genet. 9, 75–79. Kennison, J.A. (1995). The Polycomb and trithorax group proteins of Drosophila: trans-regulators of homeotic gene function. Annu. Rev. Genet. 29, 289–303. Kennison, J.A. and Russell, M.A. (1987). Dosagedependent modiﬁers of homoeotic mutations in Drosophila melanogaster. Genetics 116, 75–86. Kenyon, C. (1994). If birds can ﬂy, why can’t we? Homeotic genes and evolution. Cell 78, 175–180. Kenyon, C. (1995). A perfect vulva every time: gradients and signaling cascades in C. elegans. Cell 82, 171–174. Kenyon, K.L., Clouser, C.R., and Pignoni, F. (2001). Modulation of Sine oculis function by direct interaction with different co-factors. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a194. Keplinger, B.L., Guo, X., Quine, J., Feng, Y., and Cavener, D.R. (2001). Complex organization of promoter and enhancer elements regulate the tissue- and developmental stage-speciﬁc expression of the Drosophila melanogaster Gld gene. Genetics 157, 699–716. Kerber, B., Monge, I., Mueller, M., Mitchell, P.J., and Cohen, S.M. (2001). The AP-2 transcription factor is required for joint formation and cell survival in Drosophila leg development. Development 128, 1231–1238. Kernan, M., Cowan, D., and Zuker, C. (1994). Genetic dissection of mechanosensory transduction: mechanoreception-defective mutations of Drosophila. Neuron 12, 1195–1206. Kerppola, T. and Curran, T. (1995). Zen and the art of Fos and Jun. Nature 373, 199–200. Kerridge, S. and Morata, G. (1982). Developmental effects of some newly induced Ultrabithorax alleles of Drosophila. J. Embryol. Exp. Morph. 68, 211–234. Kerszberg, M. (1999). Morphogen propagation and action: Towards molecular models. Sems. Cell Dev. Biol. 10, 297–302. Kerszberg, M. and Changeux, J.-P. (1994). A model for

REFERENCES

2201. 2202.

2203.

2204.

2205.

2206.

2207.

2208.

2209.

2210.

2211.

2212.

2213.

2214.

2215. 2216.

2217.

reading morphogenetic gradients: autocatalysis and competition at the gene level. Proc. Natl. Acad. Sci. USA 91, 5823–5827. Kerszberg, M. and Changeux, J.-P. (1994). Partners make patterns in morphogenesis. Curr. Biol. 4, 1046–1047. Keyse, S.M. (2000). Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Curr. Opin. Cell Biol. 12, 186–192. Khalsa, O., Yoon, J.-w., Torres-Schumann, S., and Wharton, K.A. (1998). TGF-β/BMP superfamily members, Gbb-60A and Dpp, cooperate to provide pattern information and establish cell identity in the Drosophila wing. Development 125, 2723–2734. [See also 2001 Development 128, 3913–3925.] Khare, N. and Baumgartner, S. (2000). Dally-like protein, a new Drosophila glypican with expression overlapping with wingless. Mechs. Dev. 99, 199–202. Kharitonenkov, A., Chen, Z., Sures, I., Wang, H., Schilling, J., and Ullrich, A. (1997). A family of proteins that inhibit signalling through tyrosine kinase receptors. Nature 386, 181–186. Khokhlatchev, A.V., Canagarajah, B., Wilsbacher, J., Robinson, M., Atkinson, M., Goldsmith, E., and Cobb, M.H. (1998). Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation. Cell 93, 605–615. Khorasanizadeh, S. and Rastinejad, F. (1999). Transcription factors: the right combination for the DNA lock. Curr. Biol. 9, R456–R458. Kidd, S. (1992). Characterization of the Drosophila cactus locus and analysis of interactions between cactus and dorsal proteins. Cell 71, 623–635. Kidd, S., Baylies, M.K., Gasic, G.P., and Young, M.W. (1989). Structure and distribution of the Notch protein in developing Drosophila. Genes Dev. 3, 1113–1129. Kidd, S., Kelley, M.R., and Young, M.W. (1986). Sequence of the Notch locus of Drosophila melanogaster: relationship of the encoded protein to mammalian clotting and growth factors. Mol. Cell. Biol. 6, 3094–3108. Kidd, S., Lieber, T., and Young, M.W. (1998). Ligandinduced cleavage and regulation of nuclear entry of Notch in Drosophila melanogaster embryos. Genes Dev. 12, 3728– 3740. Kidwell, M.G. and Lisch, D.R. (2001). Transposable elements, parasitic DNA, and genome evolution. Evolution 55, 1–24. [See also 2001 Nature 411, 146–149.] Kiehle, C.P. and Schubiger, G. (1985). Cell proliferation changes during pattern regulation in imaginal leg discs of Drosophila melanogaster. Dev. Biol. 109, 336– 346. Kiger, J.A., Jr., Eklund, J.L., Younger, S.H., and O’Kane, C.J. (1999). Transgenic inhibitors identify two roles for protein kinase A in Drosophila development. Genetics 152, 281– 290. Kilbey, B.J. (1995). Charlotte Auerbach (1899–1994). Genetics 141, 1–5. Kim, J., Irvine, K.D., and Carroll, S.B. (1995). Cell recognition, signal induction, and symmetrical gene activation at the dorsal-ventral boundary of the developing Drosophila wing. Cell 82, 795–802. Kim, J., Johnson, K., Chen, H.J., Carroll, S., and Laughon, A. (1997). Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic. Nature 388, 304–308.

367

2218. Kim, J., Magee, J., and Carroll, S.B. (1997). Intercompartmental signaling and the regulation of vestigial expression at the dorsoventral boundary of the developing Drosophila wing. Cold Spr. Harb. Symp. Quant. Biol. 62, 283–291. 2219. Kim, J., Sebring, A., Esch, J.J., Kraus, M.E., Vorwerk, K., Magee, J., and Carroll, S.B. (1996). Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 382, 133–138. 2220. Kim, L. and Kimmel, A.R. (2000). GSK3, a master switch regulating cell-fate speciﬁcation and tumorigenesis. Curr. Opin. Gen. Dev. 10, 508–514. 2221. Kim, M.-S., Repp, A., and Smith, D.P. (1998). LUSH odorant-binding protein mediates chemosensory responses to alcohols in Drosophila melanogaster. Genetics 150, 711–721. 2222. Kimble, J. and Simpson, P. (1997). The LIN-12/Notch signaling pathway and its regulation. Annu. Rev. Cell Dev. Biol. 13, 333–361. 2223. Kimmel, B.E., Heberlein, U., and Rubin, G.M. (1990). The homeo domain protein rough is expressed in a subset of cells in the developing Drosophila eye where it can specify photoreceptor cell subtype. Genes Dev. 4, 712– 727. 2224. Kimmel, C.B. (1996). Was Urbilateria segmented? Trends Genet. 12, 329–331. 2225. Kimura, K.-i., Usui-Ishihara, A., and Usui, K. (1997). G2 arrest of cell cycle ensures a determination process of sensory mother cell formation in Drosophila. Dev. Genes Evol. 207, 199–202. 2226. Kingsley, D.M. (1994). The TGF-β superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev. 8, 133–146. 2227. Kingston, R.E., Bunker, C.A., and Imbalzano, A.N. (1996). Repression and activation by multiprotein complexes that alter chromatin structure. Genes Dev. 10, 905–920. 2228. Kingston, R.E. and Narlikar, G.J. (1999). ATP-dependent remodeling and acetylation as regulators of chromatin ﬂuidity. Genes Dev. 13, 2339–2352. 2229. Kirby, B.S. and Bryant, P.J. (1982). Growth is required for cell competition in the imaginal discs of Drosophila melanogaster. W. Roux’s Arch. 191, 289–291. 2230. Kirby, B.S., Bryant, P.J., and Schneiderman, H.A. (1982). Regeneration following duplication in imaginal wing disc fragments of Drosophila melanogaster. Dev. Biol. 90, 259– 271. 2231. Kirchhamer, C.V., Yuh, C.-H., and Davidson, E.H. (1996). Modular cis-regulatory organization of developmentally expressed genes: two genes transcribed territorially in the sea urchin embryo, and additional examples. Proc. Natl. Acad. Sci. USA 93, 9322–9328. 2232. Kirchhausen, T. (1998). Vesicle formation: Dynamic dynamin lives up to its name. Curr. Biol. 8, R792–R794. 2233. Kirkpatrick, H., Johnson, K., and Laughon, A. (2001). Repression of Dpp targets by binding of Brinker to mad sites. J. Biol. Chem. 276 #21, 18216–18222. 2234. Kirschfeld, K. (1967). Die Projektion der optischen Umwelt auf das Raster der Rhabdomere im Komplexauge von MUSCA. Exp. Brain Res. 3, 248–270. 2235. Kirschfeld, K., Feiler, R., and Franceschini, N. (1978). A photostable pigment within the rhabdomere of ﬂy photoreceptors no. 7. J. Comp. Physiol. 125, 275–284. 2236. Kirschner, M. (1992). Evolution of the cell. In “Molds, Molecules, and Metazoa: Growing Points in Evolutionary

368

2237. 2238.

2239.

2240.

2241.

2242.

2243. 2244. 2245.

2246.

2247.

2248.

2249.

2250.

2251.

2252.

2253.

REFERENCES

Biology,” (P.R. Grant and H.S. Horn, eds.). Princeton Univ. Pr.: Princeton, pp. 99–126. Kirschner, M., Gerhart, J., and Mitchison, T. (2000). Molecular “vitalism”. Cell 100, 79–88. Kishida, S., Yamamoto, H., Hino, S.-i., Ikeda, S., Kishida, M., and Kikuchi, A. (1999). DIX domains of Dvl and Axin are necessary for protein interactions and their ability to regulate β-catenin stability. Mol. Cell. Biol. 19, 4414–4422. Kishida, S., Yamamoto, H., Ikeda, S., Kishida, M., Sakamoto, I., Koyama, S., and Kikuchi, A. (1998). Axin, a negative regulator of the Wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of β-catenin. J. Biol. Chem. 273 #18, 10823–10826. Kissinger, C.R., Liu, B., Martin-Blanco, E., Kornberg, T.B., and Pabo, C.O. (1990). Crystal structure of an en˚ resolution: grailed homeodomain-DNA complex at 2.8 A a framework for understanding homeodomain-DNA interactions. Cell 63, 579–590. Kitagawa, M., Hatakeyama, S., Shirane, M., Matsumoto, M., Ishida, N., Hattori, K., Nakamichi, I., Kikuchi, A., Nakayama, K.-i., and Nakayama, K. (1999). An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of β-catenin. EMBO J. 18, 2401–2410. Kl¨ambt, C. (1993). The Drosophila gene pointed encodes two ETS-like proteins which are involved in the development of the midline glial cells. Development 117, 163– 176. Kl¨ambt, C. (1997). Drosophila development: a receptor for ommatidial recruitment. Curr. Biol. 7, R132–R135. Kl¨ambt, C. (2000). EGF receptor signalling: The importance of presentation. Curr. Biol. 10, R388–R391. Kl¨ambt, C., Glazer, L., and Shilo, B.-Z. (1992). breathless, a Drosophila FGF receptor homolog, is essential for migration of tracheal and speciﬁc midline glial cells. Genes Dev. 6, 1668–1678. Kl¨ambt, C., Knust, E., Tietze, K., and Campos-Ortega, J.A. (1989). Closely related transcripts encoded by the neurogenic gene complex Enhancer of split of Drosophila melanogaster. EMBO J. 8, 203–210. ¨ ¨ Kl¨ambt, C., Muller, S., Lutzelschwab, R., Rossa, R., Totzke, F., and Schmidt, O. (1989). The Drosophila melanogaster l(2)gl gene encodes a protein homologous to the cadherin cell-adhesion molecule family. Dev. Biol. 133, 425–436. Klein, T., Brennan, K., and Martinez Arias, A. (1997). An intrinsic dominant negative activity of Serrate that is modulated during wing development in Drosophila. Dev. Biol. 189, 123–134. Klein, T. and Campos-Ortega, J.A. (1992). Second-site modiﬁers of the Delta wing phenotype in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 202, 49–60. Klein, T. and Campos-Ortega, J.A. (1997). klumpfuss, a Drosophila gene encoding a member of the EGR family of transcription factors, is involved in bristle and leg development. Development 124, 3123–3134. Klein, T., Couso, J.P., and Martinez Arias, A. (1998). Wing development and speciﬁcation of dorsal cell fates in the absence of apterous in Drosophila. Curr. Biol. 8, 417– 420. Klein, T. and Martinez Arias, A. (1998). Different spatial and temporal interactions between Notch, wingless, and vestigial specify proximal and distal pattern elements of the wing in Drosophila. Dev. Biol. 194, 196–212. Klein, T. and Martinez Arias, A. (1998). Interactions among Delta, Serrate and Fringe modulate Notch activity during

2254.

2255.

2256.

2257.

2258.

2259.

2260.

2261. 2262.

2263.

2264.

2265.

2266.

2267.

2268.

2269.

2270.

2271.

Drosophila wing development. Development 125, 2951– 2962. Klein, T. and Martinez Arias, A. (1999). The Vestigial gene product provides a molecular context for the interpretation of signals during the development of the wing in Drosophila. Development 126, 913–925. Klein, T., Seugnet, L., Haenlin, M., and Martinez Arias, A. (2000). Two different activities of Suppressor of Hairless during wing development in Drosophila. Development 127, 3553–3566. Klemm, J.D., Beals, C.R., and Crabtree, G.R. (1997). Rapid targeting of nuclear proteins to the cytoplasm. Curr. Biol. 7, 638–644. Klemm, J.D. and Pabo, C.O. (1996). Oct-1 POU domainDNA interactions: cooperative binding of isolated subdomains and effects of covalent linkage. Genes Dev. 10, 27–36. Klemm, J.D., Rould, M.A., Aurora, R., Herr, W., and Pabo, C.O. (1994). Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules. Cell 77, 21–32. Klingenberg, C.P. and Nijhout, H.F. (1999). Genetics of ﬂuctuating asymmetry: a developmental model of developmental instability. Evolution 53, 358–375. Klingenberg, C.P. and Zaklan, S.D. (2000). Morphological integration between developmental compartments in the Drosophila wing. Evolution 54, 1273–1285. Klingensmith, J. and Nusse, R. (1994). Signaling by wingless in Drosophila. Dev. Biol. 166, 396–414. Klingensmith, J., Nusse, R., and Perrimon, N. (1994). The Drosophila segment polarity gene dishevelled encodes a novel protein required for response to the wingless signal. Genes Dev. 8, 118–130. Klueg, K.M. and Muskavitch, M.A.T. (1999). Ligandreceptor interactions and trans-endocytosis of Delta, Serrate and Notch: members of the Notch signalling pathway in Drosophila. J. Cell Sci. 112, 3289–3297. Klueg, K.M., Parody, T.R., and Muskavitch, M.A.T. (1998). Complex proteolytic processing acts on Delta, a transmembrane ligand for Notch, during Drosophila development. Mol. Biol. Cell 9, 1709–1723. Klug, A. and Schwabe, J.W.R. (1995). Zinc ﬁngers. FASEB J. 9, 597–604. [See also 2001 Annu. Rev. Biochem 70, 313–340.] Kmita, M., van der Hoeven, F., Z´ak´any, J., Krumlauf, R., and Duboule, D. (2000). Mechanisms of Hox gene colinearity: transposition of the anterior Hoxb1 gene into the posterior HoxD complex. Genes Dev. 14, 198–211. Knoblich, J.A., Jan, L.Y., and Jan, Y.N. (1995). Asymmetric segregation of Numb and Prospero during cell division. Nature 377, 624–627. Knoblich, J.A., Jan, L.Y., and Jan, Y.N. (1997). Asymmetric segregation of the Drosophila Numb protein during mitosis: facts and speculations. Cold Spr. Harb. Symp. Quant. Biol. 62, 71–77. Knoblich, J.A., Jan, L.Y., and Jan, Y.N. (1997). The N terminus of the Drosophila Numb protein directs membrane association and actin-dependent asymmetric localization. Proc. Natl. Acad. Sci. USA 94, 13005–13010. Knoblich, J.A. and Lehner, C.F. (1993). Synergistic action of Drosophila cyclins A and B during the G2 -M transition. EMBO J. 12, 65–74. Knoepﬂer, P.S. and Eisenman, R.N. (1999). Sin meets NuRD and other tails of repression. Cell 99, 447–450.

REFERENCES

2272. Knoll, A.H. and Carroll, S.B. (1999). Early animal evolution: emerging views from comparative biology and geology. Science 284, 2129–2137. 2273. Knust, E. and Leptin, M. (1996). Adherens junctions in the Drosophila embryo: the role of E-cadherin in their establishment and morphogenetic function. BioEssays 18, 609–612. 2274. Knust, E., Schrons, H., Grawe, F., and Campos-Ortega, J.A. (1992). Seven genes of the Enhancer of split Complex of Drosophila melanogaster encode helix-loop-helix proteins. Genetics 132, 505–518. 2275. Koch, C. (1997). Computation and the single neuron. Nature 385, 207–210. 2276. Koch, C. (1999). “Biophysics of Computation: Information Processing in Single Neurons.” Oxford Univ. Pr., New York. 2277. Koch, C. and Laurent, G. (1999). Complexity and the nervous system. Science 284, 96–98. 2278. Koch, P.B., Lorenz, U., Brakeﬁeld, P.M., and ffrenchConstant, R.H. (2000). Butterﬂy wing pattern mutants: developmental heterochrony and co-ordinately regulated phenotypes. Dev. Genes Evol. 210, 536–544. ¨ 2279. Kockel, L., Vorbruggen, G., J¨ackle, H., Mlodzik, M., and Bohmann, D. (1997). Requirement for Drosophila 14-33ζ in Raf-dependent photoreceptor development. Genes Dev. 11, 1140–1147. 2280. Kockel, L., Zeitlinger, J., Staszewski, L.M., Mlodzik, M., and Bohmann, D. (1997). Jun in Drosophila development: redundant and nonredundant functions and regulation by two MAPK signal transduction pathways. Genes Dev. 11, 1748–1758. 2281. Kockel, L., Zeitlinger, J., Staszewski, L.M., Mlodzik, M., and Bohmann, D. (1998). Erratum. [Kockel et al.’s 1997 ﬁnding of a synergistic interaction between dJun and dMAPK was not reproducible.]. Genes Dev. 12, 447. 2282. Koelle, M.R. (1997). A new family of G-protein regulators– the RGS proteins. Curr. Opin. Cell Biol. 9, 143–147. 2283. Kohler, R.E. (1994). “Lords of the Fly: Drosophila Genetics and the Experimental Life.” Univ. Chicago Pr., Chicago. 2284. Kohn, K.W. (1999). Molecular interaction map of the mammalian cell cycle control and DNA repair systems. Mol. Biol. Cell 10, 2703–2734. 2285. Koizumi, K., Stivers, C., Brody, T., Zangeneh, S., Mozer, B., and Odenwald, W.F. (2001). A search for Drosophila neural precursor genes identiﬁes ran. Dev. Genes Evol. 211, 67– 75. 2286. Kojima, T., Ishimaru, S., Higashijima, S.-i., Takayama, E., Akimaru, H., Sone, M., Emori, Y., and Saigo, K. (1991). Identiﬁcation of a different-type homeobox gene, BarH1, possibly causing Bar (B) and Om(1D) mutations in Drosophila. Proc. Natl. Acad. Sci. USA 88, 4343–4347. 2287. Kojima, T., Sato, M., and Saigo, K. (2000). Formation and speciﬁcation of distal leg segments in Drosophila by dual Bar homeobox genes, BarH1 and BarH2. Development 127, 769–778. 2288. Kolodkin, A.L., Pickup, A.T., Lin, D.M., Goodman, C.S., and Banerjee, U. (1994). Characterization of Star and its interactions with sevenless and EGF receptor during photoreceptor cell development in Drosophila. Development 120, 1731–1745. 2289. Komachi, K., Redd, M.J., and Johnson, A.D. (1994). The WD repeats of Tup1 interact with the homeo domain protein α2. Genes Dev. 8, 2857–2867. 2290. Kon, C.Y., Lopes da Silva, S., Cadigan, K., and Nusse, R. (2001). Tartaruga, a histone deacetylase complex member

369

2291.

2292. 2293.

2294.

2295.

2296.

2297.

2298. 2299.

2300.

2301.

2302.

2303.

2304. 2305.

2306.

2307.

2308.

which may play a role in wingless signalling. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a87. Kondo, S. (1992). A mechanistic model for morphogenesis and regeneration of limbs and imaginal discs. Mechs. Dev. 39, 161–170. Kondo, T. and Duboule, D. (1999). Breaking colinearity in the mouse HoxD complex. Cell 97, 407–417. Kondo, T. and Raff, M. (2000). Basic helix-loop-helix proteins and the timing of oligodendrocyte differentiation. Development 127, 2989–2998. Kondo, T. and Raff, M. (2000). The Id4 HLH protein and the timing of oligodendrocyte differentiation. EMBO J. 19, 1998–2007. Kondo, T., Z´ak´any, J., and Duboule, D. (1998). Control of colinearity in AbdB genes of the mouse HoxD complex. Molec. Cell 1, 289–300. Kooh, P.J., Fehon, R.G., and Muskavitch, M.A.T. (1993). Implications of dynamic patterns of Delta and Notch expression for cellular interactions during Drosophila development. Development 117, 493–507. Kooyman, D.L., Byrne, G.W., McClellan, S., Nielsen, D., Tone, M., Waldmann, H., Coffman, T.M., McCurry, K.R., Platt, J.L., and Logan, J.S. (1995). In vivo transfer of GPIlinked complement restriction factors from erythrocytes to the endothelium. Science 269, 89–92. Kopan, R. and Cagan, R. (1997). Notch on the cutting edge. Trends Genet. 13, 465–467. Kopan, R., Schroeter, E.H., Weintraub, H., and Nye, J.S. (1996). Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain. Proc. Natl. Acad. Sci. USA 93, 1683– 1688. Kopczynski, C.C., Alton, A.K., Fechtel, K., Kooh, P.J., and Muskavitch, M.A.T. (1988). Delta, a Drosophila neurogenic gene, is transcriptionally complex and encodes a protein related to blood coagulation factors and epidermal growth factor of vertebrates. Genes Dev. 2, 1723–1735. Kopp, A., Blackman, R.K., and Duncan, I. (1999). Wingless, Decapentaplegic and EGF receptor signaling pathways interact to specify dorso-ventral pattern in the adult abdomen of Drosophila. Development 126, 3495– 3507. Kopp, A. and Duncan, I. (1997). Control of cell fate and polarity in the adult abdominal segments of Drosophila by optomotor-blind. Development 124, 3715–3726. Kopp, A., Muskavitch, M.A.T., and Duncan, I. (1997). The roles of hedgehog and engrailed in patterning adult abdominal segments of Drosophila. Development 124, 3703– 3714. Kornberg, R.D. (1999). Eukaryotic transcriptional control. Trends Genet. 15, M46–M49. Kornberg, R.D. and Lorch, Y. (1999). Twenty-ﬁve years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell 98, 285–294. Kornberg, T. (1981). engrailed: A gene controlling compartment and segment formation in Drosophila. Proc. Natl. Acad. Sci. USA 78, 1095–1099. Kornberg, T., Sid´en, I., O’Farrell, P., and Simon, M. (1985). The engrailed locus of Drosophila: in situ localization of transcripts reveals compartment-speciﬁc expression. Cell 40, 45–53. Kornfeld, K., Saint, R.B., Beachy, P.A., Harte, P.J., Peattie, D.A., and Hogness, D.S. (1989). Structure and expression of a family of Ultrabithorax mRNAs generated by

370

2309.

2310. 2311.

2312.

2313.

2314.

2315.

2316.

2317.

2318. 2319.

2320.

2321.

2322.

2323.

2324.

2325.

2326.

REFERENCES

alternative splicing and polyadenylation in Drosophila. Genes Dev. 3, 243–258. Korzh, V. and Grunwald, D. (2001). Nadine Dobrovolska¨ıa-Zavadska¨ıa and the dawn of developmental genetics. BioEssays 23, 365–371. Koshland, D.E., Jr. (1998). The era of pathway quantiﬁcation. Science 280, 852–853. Koshland, D.E., Jr., Goldbeter, A., and Stock, J.B. (1982). Ampliﬁcation and adaptation in regulatory and sensory systems. Science 217, 220–225. Koushika, S.P., Lisbin, M.J., and White, K. (1996). ELAV, a Drosophila neuron-speciﬁc protein, mediates the generation of an alternatively spliced neural protein isoform. Curr. Biol. 12, 1634–1641. [See also 2001 Genes Dev. 15, 2546–2561.] Koushika, S.P., Soller, M., and White, K. (2000). The neuron-enriched splicing pattern of Drosophila erect wing is dependent on the presence of ELAV protein. Mol. Cell. Biol. 20, 1836–1845. Kozmik, Z., Czerny, T., and Busslinger, M. (1997). Alternatively spliced insertions in the paired domain restrict the DNA sequence speciﬁcity of Pax6 and Pax8. EMBO J. 16, 6793–6803. Kozopas, K.M., Samos, C.H., and Nusse, R. (1998). DWnt2, a Drosophila Wnt gene required for the development of the male reproductive tract, speciﬁes a sexually dimorphic cell fate. Genes Dev. 12, 1155–1165. Krakauer, D.C. and Nowak, M.A. (1999). Evolutionary preservation of redundant duplicated genes. Sems. Cell Dev. Biol. 10, 555–559. Kramatschek, B. and Campos-Ortega, J.A. (1994). Neuroectodermal transcription of the Drosophila neurogenic genes E(spl) and HLH-m5 is regulated by proneural genes. Development 120, 815–826. Kr¨amer, H. (1993). Patrilocal cell-cell interactions: sevenless captures its bride. Trends Cell Biol. 3, 103–105. Kr¨amer, H., Cagan, R.L., and Zipursky, S.L. (1991). Interaction of bride of sevenless membrane-bound ligand and the sevenless tyrosine-kinase receptor. Nature 352, 207– 212. Kr¨amer, H. and Phistry, M. (1999). Genetic analysis of hook, a gene required for endocytic trafﬁcking in Drosophila. Genetics 151, 675–684. Kramer, S., Okabe, M., Hacohen, N., Krasnow, M.A., and Hiromi, Y. (1999). Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila. Development 126, 2515–2525. Kramer, S., West, S.R., and Hiromi, Y. (1995). Cell fate control in the Drosophila retina by the orphan receptor sevenup: its role in the decisions mediated by the ras signaling pathway. Development 121, 1361–1372. Krantz, D.E. and Zipursky, S.L. (1990). Drosophila chaoptin, a member of the leucine-rich repeat family, is a photoreceptor cell-speciﬁc adhesion molecule. EMBO J. 9, 1969–1977. Krasnow, M.A., Saffman, E.E., Kornfeld, K., and Hogness, D.S. (1989). Transcriptional activation and repression by Ultrabithorax proteins in cultured Drosophila cells. Cell 57, 1031–1043. Krasnow, R.E. and Adler, P.N. (1994). A single frizzled protein has a dual function in tissue polarity. Development 120, 1883–1893. Krasnow, R.E., Wong, L.L., and Adler, P.N. (1995). dishevelled is a component of the frizzled signaling pathway in Drosophila. Development 121, 4095–4102.

2327. Kraut, R. and Campos-Ortega, J.A. (1996). inscuteable, a neural precursor gene of Drosophila, encodes a candidate for a cytoskeleton adaptor protein. Dev. Biol. 174, 65–81. 2328. Kraut, R., Chia, W., Jan, L.Y., Jan, Y.N., and Knoblich, J.A. (1996). Role of inscuteable in orienting asymmetric cell divisions in Drosophila. Nature 383, 50–55. 2329. Kraut, R. and Levine, M. (1991). Mutually repressive interactions between the gap genes giant and Kruppel deﬁne ¨ middle body regions of the Drosophila embryo. Development 111, 611–621. 2330. Krebs, J.E. and Dunaway, M. (1998). The scs and scs insulator elements impart a cis requirement on enhancerpromoter interactions. Molec. Cell 1, 301–308. 2331. Kretzschmar, D., Brunner, A., Wiersdorff, V., Pﬂugfelder, G.O., Heisenberg, M., and Schneuwly, S. (1992). giant lens, a gene involved in cell determination and axon guidance in the visual system of Drosophila melanogaster. EMBO J. 11, 2531–2539. 2332. Kretzschmar, M. and Massagu´e, J. (1998). SMADs: mediators and regulators of TGF-β signaling. Curr. Opin. Gen. Dev. 8, 103–111. 2333. Krishnan, B., Dryer, S.E., and Hardin, P.E. (1999). Circadian rhythms in olfactory responses of Drosophila melanogaster. Nature 400, 375–378. 2334. Krylov, D., Mikhailenko, I., and Vinson, C. (1994). A thermodynamic scale for leucine zipper stability and dimerization speciﬁcity: e and g interhelical interactions. EMBO J. 13, 2849–2861. 2335. Kubota, K., Goto, S., Eto, K., and Hayashi, S. (2000). EGF receptor attenuates Dpp signaling and helps to distinguish the wing and leg cell fates in Drosophila. Development 127, 3769–3776. 2336. Kucharczuk, K.L., Love, C.M., Dougherty, N.M., and Goldhamer, D.J. (1999). Fine-scale transgenic mapping of the MyoD core enhancer: MyoD is regulated by distinct but overlapping mechanisms in myotomal and non-myotomal muscle lineages. Development 126, 1957– 1965. ¨ 2337. Kuhl, M. and Wedlich, D. (1997). Wnt signalling goes nuclear. BioEssays 19, 101–104. ¨ 2338. Kuhn, A. (1971). “Lectures on Developmental Physiology.” 2nd ed. Springer-Verlag, Berlin. 2339. Kuhn, D.T. and Cunningham, G.N. (1977). Aldehyde oxidase compartmentalization in Drosophila melanogaster wing imaginal disks. Science 196, 875–877. 2340. Kuhn, D.T. and Cunningham, G.N. (1978). Aldehyde oxidase distribution in Drosophila melanogaster mature imaginal discs, histoblasts and rings of imaginal cells. J. Exp. Zool. 204, 1–9. 2341. Kuhn, D.T. and Cunningham, G.N. (1986). Isocitrate dehydrogenase in D. melanogaster imaginal discs: Pattern development and alteration by homoeotic mutant genes. Dev. Genet. 7, 21–34. 2342. Kuhn, D.T., Fogerty, S.C., Eskens, A.A.C., and Sprey, T.E. (1983). Developmental compartments in the Drosophila melanogaster wing disc. Dev. Biol. 95, 399–413. 2343. Kuhn, D.T. and Freeland, D.E. (1996). Expression patterns of developmental genes reveal segment and parasegment organization of D. melanogaster genital discs. Mechs. Dev. 56, 61–72. 2344. Kuhn, D.T. and Sprey, T.E. (1987). Regulation of NADPmalic enzyme in the eye-antennal imaginal disc of D. melanogaster/D. simulans hybrids: Evidence for cis- and trans- regulation. Genetics 115, 277–281. ¨ 2345. Kuhnlein, R.P., Br¨onner, G., Taubert, H., and Schuh, R.

REFERENCES

2346.

2347.

2348.

2349.

2350.

2351.

2352.

2353.

2354.

2355.

2356.

2357. 2358.

2359.

2360.

2361.

(1997). Regulation of Drosophila spalt gene expression. Mechs. Dev. 66, 107–118. ¨ Kuhnlein, R.P., Frommer, G., Friedrich, M., GonzalezGaitan, M., Weber, A., Wagner-Bernholz, J.F., Gehring, W.J., J¨ackle, H., and Schuh, R. (1994). spalt encodes an evolutionarily conserved zinc ﬁnger protein of novel structure which provides homeotic gene function in the head and tail region of the Drosophila embryo. EMBO J. 13, 168–179. ¨ Kuhnlein, R.P. and Schuh, R. (1996). Dual function of the region-speciﬁc homeotic gene spalt during Drosophila tracheal system development. Development 122, 2215– 2223. Kukalov´a-Peck, J. (1983). Origin of the insect wing and wing articulation from the arthropodan leg. Can. J. Zool. 61, 1618–1669. Kukalov´a-Peck, J. (1985). Ephemeroid wing venation based upon new gigantic Carboniferous mayﬂies and basic morphology, phylogeny, and metamorphosis of pterygote insects (Insecta, Ephemerida). Can. J. Zool. 63, 933–955. Kulkarni, M.M. and Arnosti, D.N. (2001). In vivo characterization of short-range transcriptional repression. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a124. Kumar, J. and Moses, K. (1997). Transcription factors in eye development: a gorgeous mosaic? Genes Dev. 11, 2023– 2028. Kumar, J.P. and Moses, K. (2000). Cell fate speciﬁcation in the Drosophila retina. In “Vertebrate Eye Development,” Results and Problems in Cell Differentiation, Vol. 31 (M.E. Fini, ed.). Springer: Berlin, pp. 93–114. Kumar, J.P. and Moses, K. (2001). EGF receptor and Notch signaling act upstream of Eyeless/Pax6 to control eye speciﬁcation. Cell 104, 687–697. [See also 2001 references: Development 128, 2689–2697; Dev. Genes Evol. 211, 406–414; Nature Rev. Genet. 2, 846–857; Sems. Cell Dev. Biol. 12, 469–474.] Kumar, J.P. and Ready, D.F. (1995). Rhodopsin plays an essential structural role in Drosophila photoreceptor development. Development 121, 4359–4370. Kumar, J.P., Tio, M., Hsiung, F., Akopyan, S., Gabay, L., Seger, R., Shilo, B.-Z., and Moses, K. (1998). Dissecting the roles of the Drosophila EGF receptor in eye development and MAP kinase activation. Development 125, 3875–3885. Kuner, J.M., Nakanishi, M., Ali, Z., Drees, B., Gustavson, E., Theis, J., Kauvar, L., Kornberg, T., and O’Farrell, P.H. (1985). Molecular cloning of engrailed: a gene involved in the development of pattern in Drosophila melanogaster. Cell 42, 309–316. Kunes, S. (1999). Stop or Go in the target zone. Neuron 22, 639–640. Kunes, S. and Steller, H. (1991). Ablation of Drosophila photoreceptor cells by conditional expression of a toxin gene. Genes Dev. 5, 970–983. Kunisch, M., Haenlin, M., and Campos-Ortega, J.A. (1994). Lateral inhibition mediated by the Drosophila neurogenic gene Delta is enhanced by proneural proteins. Proc. Natl. Acad. Sci. USA 91, 10139–10143. Kurant, E., Eytan, D., and Salzberg, A. (2001). Mutational analysis of the Drosophila homothorax gene. Genetics 157, 689–698. Kurant, E., Pai, C.-y., Sharf, R., Halachmi, N., Sun, Y.H., and Salzberg, A. (1998). dorsotonals/homothorax, the Drosophila homologue of meis1, interacts with extradenticle in patterning of the embryonic PNS. Development 125, 1037–1048.

371

2362. Kurata, S., Go, M.J., Artavanis-Tsakonas, S., and Gehring, W.J. (2000). Notch signaling and the determination of appendage identity. Proc. Natl. Acad. Sci. USA 97, 2117– 2122. 2363. Kuriyama, M., Harada, N., Kuroda, S., Yamamoto, T., Nakafuku, M., Iwamatsu, A., Yamamoto, D., Prasad, R., Croce, C., Canaani, E., and Kaibuchi, K. (1996). Identiﬁcation of AF-6 and Canoe as putative targets for Ras. J. Biol. Chem. 271 #2, 607–610. 2364. Kuriyan, J. and Cowburn, D. (1997). Modular peptide recognition domains in eukaryotic signaling. Annu. Rev. Biophys. Biomol. Struct. 26, 259–288. 2365. Kuriyan, J. and Darnell, J.E., Jr. (1999). An SH2 domain in disguise. Nature 398, 22–25. 2366. Kurokawa, M., Tanaka, T., Tanaka, K., Hirano, N., Ogawa, S., Mitani, K., Yazaki, Y., and Hirai, H. (1996). A conserved cysteine residue in the runt homology domain of AML1 is required for the DNA binding ability and the transforming activity on ﬁbroblasts. J. Biol. Chem. 271 #28, 16870– 16876. 2367. Kurusu, M., Nagao, T., Walldorf, U., Flister, S., Gehring, W.J., and Furukubo-Tokunaga, K. (2000). Genetic control of development of the mushroom bodies, the associative learning centers in the Drosophila brain, by the eyeless, twin of eyeless, and dachshund genes. Proc. Natl. Acad. Sci. USA 97, 2140–2144. ¨ 2368. Kussel-Andermann, P., El-Amraoui, A., Saﬁeddine, S., Nouaille, S., Perfettini, I., Lecuit, M., Cossart, P., Wolfrum, U., and Petit, C. (2000). Vezatin, a novel transmembrane protein, bridges myosin VIIA to the cadherin-catenins complex. EMBO J. 19, 6020–6029. 2369. Kutty, G., Kutty, R.K., Samuel, W., Duncan, T., Jaworski, C., and Wiggert, B. (1998). Identiﬁcation of a new member of Transforming Growth Factor-Beta superfamily in Drosophila: the ﬁrst invertebrate activin gene. Biochem. Biophys. Res. Comm. 246, 644–649. 2370. Kuzin, B., Roberts, I., Peunova, N., and Enikolopov, G. (1996). Nitric oxide regulates cell proliferation during Drosophila development. Cell 87, 639–649. 2371. Kuziora, M.A. (1993). Abdominal-B protein isoforms exhibit distinct cuticular transformations and regulatory activities when ectopically expressed in Drosophila embryos. Mechs. Dev. 42, 125–137. 2372. Kuziora, M.A. and McGinnis, W. (1988). Autoregulation of a Drosophila homeotic selector gene. Cell 55, 477–485. 2373. Kyba, M. and Brock, H.W. (1998). The Drosophila Polycomb group protein Psc contacts ph and Pc through speciﬁc conserved domains. Mol. Cell. Biol. 18, 2712–2720. 2374. Kyba, M. and Brock, H.W. (1998). The SAM domain of Polyhomeotic, RAE28, and Scm mediates speciﬁc interactions through conserved residues. Dev. Genet. 22, 74–84. 2375. Kylsten, P. and Saint, R. (1997). Imaginal tissues of Drosophila melanogaster exhibit different modes of cell proliferation control. Dev. Biol. 192, 509–522. 2376. La Ros´ee, A., H¨ader, T., Taubert, H., Rivera-Pomar, R., and J¨ackle, H. (1997). Mechanism and Bicoid-dependent control of hairy stripe 7 expression in the posterior region of the Drosophila embryo. EMBO J. 16, 4403–4411. 2377. Lacalli, T.C. (1990). Modeling the Drosophila pair-rule pattern by reaction-diffusion: gap input and pattern control in a 4-morphogen system. J. Theor. Biol. 144, 171–194. 2378. Lacalli, T.C. and Harrison, L.G. (1991). From gradient to segments: models for pattern formation in early Drosophila embryogenesis. Sems. Dev. Biol. 2, 107–117. 2379. Lacalli, T.C., Wilkinson, D.A., and Harrison, L.G. (1988).

372

2380. 2381.

2382.

2383.

2384.

2385.

2386.

2387.

2388. 2389.

2390.

2391.

2392.

2393.

2394.

2395. 2396.

2397.

REFERENCES

Theoretical aspects of stripe formation in relation to Drosophila segmentation. Development 104, 105–113. Lagna, G. and Hemmati-Brivanlou, A. (1999). A molecular basis for Smad speciﬁcity. Dev. Dynamics 214, 269–277. Lai, C., Lyman, R.F., Long, A.D., Langley, C.H., and Mackay, T.F.C. (1994). Naturally occurring variation in bristle number and DNA polymorphisms at the scabrous locus of Drosophila melanogaster. Science 266, 1697–1702. Lai, E.C., Bodner, R., Kavaler, J., Freschi, G., and Posakony, J.W. (2000). Antagonism of Notch signaling activity by members of a novel protein family encoded by the Bearded and Enhancer of split gene complexes. Development 127, 291–306. Lai, E.C., Bodner, R., and Posakony, J.W. (2000). The Enhancer of split Complex of Drosophila includes four Notch-regulated members of the Bearded gene family. Development 127, 3441–3455. Lai, E.C., Burks, C., and Posakony, J.W. (1998). The K box, a conserved 3 UTR sequence motif, negatively regulates accumulation of Enhancer of split Complex transcripts. Development 125, 4077–4088. Lai, E.C. and Posakony, J.W. (1997). The Bearded box, a novel 3 UTR sequence motif, mediates negative posttranscriptional regulation of Bearded and Enhancer of split Complex gene expression. Development 124, 4847–4856. Lai, E.C. and Posakony, J.W. (1998). Regulation of Drosophila neurogenesis by RNA: RNA duplexes? Cell 93, 1103–1104. Lai, E.C. and Rubin, G.M. (2001). neuralized functions cellautonomously to regulate a subset of Notch-dependent processes during adult Drosophila development. Dev. Biol. 231, 217–233. [See also 2001 references: Dev. Cell 1, 725–731, 783–794, and 807–816; Proc. Natl. Acad. Sci. USA 98, 5637–5642.] Lai, E.p. (2000). Bristling all over. Curr. Biol. 10, R431. Lai, K.-M.V., Olivier, J.P., Gish, G.D., Henkemeyer, M., McGlade, J., and Pawson, T. (1995). A Drosophila shc gene product is implicated in signaling by the DER receptor tyrosine kinase. Mol. Cell. Biol. 15, 4810–4818. Lai, Z.-C., Fetchko, M., and Li, Y. (1997). Repression of Drosophila photoreceptor cell fate through cooperative action of two transcriptional repressors Yan and Tramtrack. Genetics 147, 1131–1137. Lai, Z.-C. and Li, Y. (1999). Tramtrack69 is positively and autonomously required for Drosophila photoreceptor development. Genetics 152, 299–305. Lai, Z.-C. and Rubin, G.M. (1992). Negative control of photoreceptor development in Drosophila by the product of the yan gene, an ETS domain protein. Cell 70, 609–620. LaJeunesse, D. and Shearn, A. (1995). Trans-regulation of thoracic homeotic selector genes of the Antennapedia and bithorax complexes by the trithorax group genes: absent, small, and homeotic discs 1 and 2. Mechs. Dev. 53, 123–139. Lake, R.J., Wakabayashi-Ito, N., and Artavanis-Tsakonas, S. (2001). Elucidating the role of Mastermind in Notch signaling. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a147. Lakin-Thomas, P.L. (2000). Circadian rhythms: new functions for old clock genes? Trends Genet. 16, 135–142. Lamka, M.L., Boulet, A.M., and Sakonju, S. (1992). Ectopic expression of UBX and ABD-B proteins during Drosophila embryogenesis: competition, not a functional hierarchy, explains phenotypic suppression. Development 116, 841– 854. Lammel, U., Meadows, L., and Saumweber, H. (2000).

2398.

2399. 2400.

2401.

2402. 2403.

2404.

2405.

2406.

2407.

2408.

2409.

2410.

2411.

2412.

2413.

2414. 2415.

2416.

2417.

Analysis of Drosophila salivary gland, epidermis and CNS development suggests an additional function of brinker in anterior-posterior cell fate speciﬁcation. Mechs. Dev. 92, 179–191. Landauer, W. (1958). On phenocopies, their developmental physiology and genetic meaning. Am. Nat. 92, 201– 213. Landauer, W. (1959). The phenocopy concept: illusion or reality? Experientia 15, 409–412. Landecker, H.L., Sinclair, D.A.R., and Brock, H.W. (1994). Screen for enhancers of Polycomb and Polycomblike in Drosophila melanogaster. Dev. Genet. 15, 425–434. Landschulz, W.H., Johnson, P.F., and McKnight, S.L. (1988). The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240, 1759– 1764. Landsman, D. and Bustin, M. (1993). A signature for the HMG-1 Box DNA-binding proteins. BioEssays 15, 539–546. Lane, M.E. and Kalderon, D. (1993). Genetic investigation of cAMP-dependent protein kinase function in Drosophila development. Genes Dev. 7, 1229–1243. Lane, M.E., Sauer, K., Wallace, K., Jan, Y.N., Lehner, C.F., and Vaessin, H. (1996). Dacapo, a cyclin-dependent kinase inhibitor, stops cell proliferation during Drosophila development. Cell 87, 1225–1235. Lanoue, B.R. and Jacobs, J.R. (1999). rhomboid function in the midline of the Drosophila CNS. Dev. Genet. 25, 321–330. Larsen, E., Lee, T., and Glickman, N. (1996). Antenna to leg transformation: dynamics of developmental competence. Dev. Genet. 19, 333–339. Larsen, E. and McLaughlin, H.M.G. (1987). The morphogenetic alphabet: lessons for simple-minded genes. BioEssays 7, 130–132. Larsen, E. and Zorn, A. (1989). Cell patterns associated with normal and mutant morphogenesis in silverimpregnated imaginal discs of Drosophila. Trans. Am. Microsc. Soc. 108, 51–57. Larsen, E.W. (1992). Tissue strategies as developmental constraints: implications for animal evolution. Trends Ecol. Evol. 7, 414–417. Larsen, E.W. (1997). Evolution of development: The shufﬂing of ancient modules by ubiquitous bureaucracies. In “Physical Theory in Biology: Foundations and Explorations,” (C.J. Lumsden, W.A. Brandts, and L.E.H. Trainor, eds.). World Scientiﬁc: Singapore, pp. 431–441. Larsen-Rapport, E.W. (1986). Imaginal disc determination: molecular and cellular correlates. Annu. Rev. Entomol. 31, 145–175. Larsson, J., Chen, J.D., Rasheva, V., Rasmuson-Lestander, A., and Pirrotta, V. (2001). Painting of fourth, a chromosome-speciﬁc protein in Drosophila. Proc. Natl. Acad. Sci. USA 98, 6273–6278. Latter, B.D.H. (1970). Selection for a threshold character in Drosophila. III. Genetic control of variability in plateaued populations. Genet. Res., Camb. 15, 285–300. Laubichler, M.D. (1999). Seeing is believing, but what do we see? Science 284, 58. Laudet, V., Stehelin, D., and Clevers, H. (1993). Ancestry and diversity of the HMG box superfamily. Nucleic Acids Res. 21, 2493–2501. Laughon, A.S., Kirkpatrick, H., and Johnson, K. (2001). Repression of Dpp targets by Brinker. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a125. Lavrov, S.A. and Cavalli, G. (2001). Chromatin organisation

REFERENCES

2418.

2419.

2420. 2421.

2422.

2423.

2424.

2425.

2426. 2427.

2428.

2429. 2430. 2431. 2432. 2433. 2434. 2435. 2436.

2437.

2438.

of the cellular memory module (CMM). Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a96. Lawler, S., Feng, X.-H., Chen, R.-H., Maruoka, E.M., Turck, C.W., Griswold-Prenner, I., and Derynck, R. (1997). The type II transforming growth factor-β receptor autophosphorylates not only on serine and threonine but also on tyrosine residues. J. Biol. Chem. 272 #23, 14850– 14859. Lawrence, N., Klein, T., Brennan, K., and Martinez Arias, A. (2000). Structural requirements for Notch signalling with Delta and Serrate during the development and patterning of the wing disc of Drosophila. Development 127, 3185–3195. Lawrence, P. (1979). Squaring the circle. Nature 280, 722– 723. Lawrence, P. and Morata, G. (1994). Homeobox genes: their function in Drosophila segmentation and pattern formation. Cell 78, 181–189. Lawrence, P.A. (1966). Development and determination of hairs and bristles in the milkweed bug Oncopeltus fasciatus (Lygaeidae, Hemiptera). J. Cell Sci. 1, 475–498. Lawrence, P.A. (1966). Gradients in the insect segment: the orientation of hairs in the milkweed bug Oncopeltus fasciatus. J. Exp. Biol. 44, 607–620. Lawrence, P.A. (1967). The insect epidermal cell–“A simple model of the embryo”. In “Insects and Physiology,” (J.W.L. Beament and J.E. Treherne, eds.). Oliver & Boyd: London, pp. 53–68. Lawrence, P.A. (1970). Polarity and patterns in the postembryonic development of insects. Adv. Insect Physiol. 7, 197– 266. Lawrence, P.A. (1971). The organization of the insect segment. Symp. Soc. Exp. Biol. 25, 379–392. Lawrence, P.A. (1973). The development of spatial patterns in the integument of insects. In “Developmental Systems: Insects,” Vol. 2 (S.J. Counce and C.H. Waddington, eds.). Acad. Pr.: New York, pp. 157–209. Lawrence, P.A. (1975). The cell cycle and cellular differentiation in insects. In “Cell Cycle and Cell Differentiation,” (J. Reinert and H. Holtzer, eds.). Springer: Berlin, pp. 111–121. Lawrence, P.A., ed. (1976). “Insect Development.” Symp. Roy. Entomol. Soc. Lond., Vol. 8. Wiley, New York. Lawrence, P.A. (1981). The cellular basis of segmentation in insects. Cell 26, 3–10. Lawrence, P.A. (1984). Homoeotic selector genes–a working deﬁnition. BioEssays 1, 227–229. Lawrence, P.A. (1985). Compartment genes in hand. Nature 313, 268–269. Lawrence, P.A. (1987). Pair-rule genes: do they paint stripes or draw lines? Cell 51, 879–880. Lawrence, P.A. (1992). “The Making of a Fly: The Genetics of Animal Design.” Blackwell Sci. Pub., Oxford. Lawrence, P.A. (1997). Straight and wiggly afﬁnities. Nature 389, 546–547. Lawrence, P.A., Casal, J., and Struhl, G. (1999). hedgehog and engrailed: pattern formation and polarity in the Drosophila abdomen. Development 126, 2431–2439. Lawrence, P.A., Casal, J., and Struhl, G. (1999). The Hedgehog morphogen and gradients of cell afﬁnity in the abdomen of Drosophila. Development 126, 2441–2449. Lawrence, P.A., Johnston, P., Macdonald, P., and Struhl, G. (1987). Borders of parasegments in Drosophila embryos are delimited by the fushi tarazu and even-skipped genes. Nature 328, 440–442.

373

2439. Lawrence, P.A. and Locke, M. (1997). A man for our season. Nature 386, 757–758. 2440. Lawrence, P.A. and Morata, G. (1976). The compartment hypothesis. In “Insect Development,” Symp. Roy. Entomol. Soc. Lond., Vol. 8 (P.A. Lawrence, ed.). Wiley: New York, pp. 132–149. 2441. Lawrence, P.A. and Morata, G. (1976). Compartments in the wing of Drosophila: A study of the engrailed gene. Dev. Biol. 50, 321–337. 2442. Lawrence, P.A. and Morata, G. (1977). The early development of mesothoracic compartments in Drosophila: An analysis of cell lineage and fate mapping and an assessment of methods. Dev. Biol. 56, 40–51. 2443. Lawrence, P.A. and Morata, G. (1979). Pattern formation and compartments in the tarsus of Drosophila. In “Determinants of Spatial Organization,” Symp. Soc. Dev. Biol., Vol. 37 (S. Subtelny and I.R. Konigsberg, eds.). Acad. Pr.: New York, pp. 317–323. 2444. Lawrence, P.A. and Morata, G. (1992). Lighting up Drosophila. Nature 356, 107–108. 2445. Lawrence, P.A. and Morata, G. (1993). A no-wing situation. Nature 366, 305–306. 2446. Lawrence, P.A. and Sampedro, J. (1993). Drosophila segmentation: after the ﬁrst three hours. Development 119, 971–976. 2447. Lawrence, P.A. and Struhl, G. (1982). Further studies of the engrailed phenotype in Drosophila. EMBO J. 1, 827–833. 2448. Lawrence, P.A. and Struhl, G. (1996). Morphogens, compartments, and pattern: lessons from Drosophila? Cell 85, 951–961. [See also 2001 Cell 105, 559–562.] 2449. Lawrence, P.A., Struhl, G., and Morata, G. (1979). Bristle patterns and compartment boundaries in the tarsi of Drosophila. J. Embryol. Exp. Morphol. 51, 195–208. 2450. Lawrence, P.A. and Tomlinson, A. (1991). A marriage is consummated. Nature 352, 193. 2451. Lear, B.C., Skeath, J.B., and Patel, N.H. (1999). Neural cell fate in rca1 and cycA mutants: the roles of intrinsic and extrinsic factors in asymmetric division in the Drosophila central nervous system. Mechs. Dev. 88, 207–219. 2452. Lebovitz, R.M. and Ready, D.F. (1986). Ommatidial development in Drosophila eye disc fragments. Dev. Biol. 117, 663–671. 2453. Lecourtois, M. and Schweisguth, F. (1995). The neurogenic Suppressor of Hairless DNA-binding protein mediates the transcriptional activation of the Enhancer of split Complex genes triggered by Notch signaling. Genes Dev. 9, 2598– 2608. 2454. Lecourtois, M. and Schweisguth, F. (1998). Indirect evidence for Delta-dependent intracellular processing of Notch in Drosophila embryos. Curr. Biol. 8, 771–774. 2455. Lecuit, T., Brook, W.J., Ng, M., Calleja, M., Sun, H., and Cohen, S.M. (1996). Two distinct mechanisms for longrange patterning by Decapentaplegic in the Drosophila wing. Nature 381, 387–393. 2456. Lecuit, T. and Cohen, S.M. (1997). Proximal-distal axis formation in the Drosophila leg. Nature 388, 139–145. 2457. Lecuit, T. and Cohen, S.M. (1998). Dpp receptor levels contribute to shaping the Dpp morphogen gradient in the Drosophila wing imaginal disc. Development 125, 4901– 4907. 2458. Lee, A., Frank, D.W., Marks, M.S., and Lemmon, M.A. (1999). Dominant-negative inhibition of receptormediated endocytosis by a dynamin-1 mutant with a defective pleckstrin homology domain. Curr. Biol. 9, 261– 264.

374

2459. Lee, C., Parikh, V., Itsukaichi, T., Bae, K., and Edery, I. (1996). Resetting the Drosophila clock by photic regulation of PER and a PER-TIM complex. Science 271, 1740– 1744. 2460. Lee, E.-C. and Baker, N.E. (1996). Gp300Sca is not a high afﬁnity Notch ligand. Biochem. Biophys. Res. Comm. 225, 720–725. 2461. Lee, E.-C., Hu, X., Yu, S.-Y., and Baker, N.E. (1996). The scabrous gene encodes a secreted glycoprotein dimer and regulates proneural development in Drosophila eyes. Molec. Cell Biol. 16, 1179–1188. 2462. Lee, E.-C., Yu, S.-Y., and Baker, N.E. (2000). The Scabrous protein can act as an extracellular antagonist of Notch signaling in the Drosophila wing. Curr. Biol. 10, 931– 934. 2463. Lee, E.-C., Yu, S.-Y., Hu, X., Mlodzik, M., and Baker, N.E. (1998). Functional analysis of the Fibrinogen-related scabrous gene from Drosophila melanogaster identiﬁes potential effector and stimulatory protein domains. Genetics 150, 663–673. 2464. Lee, J.D., Kraus, P., Gaiano, N., Nery, S., Kohtz, J., Fishell, G., Loomis, C.A., and Treisman, J.E. (2001). An acylatable residue of Hedgehog is differentially required in Drosophila and mouse limb development. Dev. Biol. 233, 122–136. 2465. Lee, J.D. and Treisman, J.E. (2001). The role of Wingless signaling in establishing the anteroposterior and dorsoventral axes of the eye disc. Development 128, 1519–1529. 2466. Lee, J.J., Ekker, S.C., von Kessler, D.P., Porter, J.A., Sun, B.I., and Beachy, P.A. (1994). Autoproteolysis in hedgehog protein biogenesis. Science 266, 1528–1537. 2467. Lee, J.J., von Kessler, D.P., Parks, S., and Beachy, P.A. (1992). Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog. Cell 71, 33–50. 2468. Lee, K.J., Freeman, M., and Steller, H. (1991). Expression of the disconnected gene during development of Drosophila melanogaster. EMBO J. 10, 817–826. 2469. Lee, T., Hacohen, N., Krasnow, M., and Montell, D.J. (1996). Regulated Breathless receptor tyrosine kinase activity required to pattern cell migration and branching in the Drosophila tracheal system. Genes Dev. 10, 2912–2921. 2470. Lee, T. and Montell, D.J. (1997). Multiple Ras signals pattern the Drosophila ovarian follicle cells. Dev. Biol. 185, 25–33. 2471. Lee, T.I., Wyrick, J.J., Koh, S.S., Jennings, E.G., Gadbois, E.L., and Young, R.A. (1998). Interplay of positive and negative regulators in transcription initiation by RNA polymerase II holoenzyme. Mol. Cell. Biol. 18, 4455–4462. 2472. Lees, A.D. (1941). Operations on the pupal wing of Drosophila melanogaster. J. Genet. 42, 115–142. 2473. Lees, A.D. (1942). Homology of the campaniform organs on the wing of Drosophila melanogaster. Nature 150, 375. 2474. Lees, A.D. and Picken, L.E.R. (1945). Shape in relation to ﬁne structure in the bristles of Drosophila melanogaster. Proc. Roy. Soc. Lond. (Ser. B.) 132, 396–423. [See also 2001 Development 128, 2793–2802.] 2475. Lees, A.D. and Waddington, C.H. (1942). The development of the bristles in normal and some mutant types of Drosophila melanogaster. Proc. Roy. Soc. (London), Ser. B 131, 87–101. 2476. Leevers, S.J. (2001). Growth control: invertebrate insulin surprises! Curr. Biol. 11, R209–R212. 2477. Leevers, S.J., Paterson, H.F., and Marshall, C.J. (1994).

REFERENCES

2478.

2479.

2480.

2481.

2482.

2483.

2484.

2485.

2486.

2487.

2488.

2489.

2490.

2491.

2492.

2493.

2494.

2495.

Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369, 411–414. Leevers, S.J., Vanhaesebroeck, B., and Waterﬁeld, M.D. (1999). Signalling through phosphoinositide 3-kinases: the lipids take center stage. Curr. Opin. Cell Biol. 11, 219– 225. Lefers, M.A., Wang, Q.T., and Holmgren, R.A. (2001). Genetic dissection of the Drosophila Cubitus interruptus signaling complex. Dev. Biol. 236, 411–420. Lehman, D.A., Patterson, B., Johnston, L.A., Balzer, T., Britton, J.S., Saint, R., and Edgar, B.A. (1999). Cis-regulatory elements of the mitotic regulator, string/Cdc25. Development 126, 1793–1803. Lehner, C.F. and O’Farrell, P.H. (1989). Expression and function of Drosophila Cyclin A during embryonic cell cycle progression. Cell 56, 957–968. Lehner, C.F. and O’Farrell, P.H. (1990). The roles of Drosophila cyclins A and B in mitotic control. Cell 61, 535– 547. Lei, Y. and Warrior, R. (2000). The Drosophila Lissencephaly1 (DLis1) gene is required for nuclear migration. Dev. Biol. 226, 57–72. Leimeister, C., Dale, K., Fischer, A., Klamt, B., Hrabe de Angelis, M., Radtke, F., McGrew, M.J., Pourqui´e, O., and Gessler, M. (2000). Oscillating expression of c-Hey2 in the presomitic mesoderm suggests that the segmentation clock may use combinatorial signaling through multiple interacting bHLH factors. Dev. Biol. 227, 91–103. Leiserson, W.M., Benzer, S., and Bonini, N.M. (1998). Dual functions of the Drosophila eyes absent gene in the eye and embryo. Mechs. Dev. 73, 193–202. Leloup, J.-C. and Goldbeter, A. (2000). Modeling the molecular regulatory mechanism of circadian rhythms in Drosophila. BioEssays 22, 84–93. Lemmon, M.A., Ferguson, K.M., and Schlessinger, J. (1996). PH domains: Diverse sequences with a common fold recruit signaling molecules to the cell surface. Cell 85, 621–624. Lemon, B. and Tjian, R. (2000). Orchestrated response: a symphony of transcription factors for gene control. Genes Dev. 14, 2551–2569. Lemon, B.D. and Freedman, L.P. (1999). Nuclear receptor cofactors as chromatin remodelers. Curr. Opin. Gen. Dev. 9, 499–504. Lemon, K.P. and Grossman, A.D. (2000). Movement of replicating DNA through a stationary replisome. Molec. Cell 6, 1321–1330. Lepage, T., Cohen, S.M., Diaz-Benjumea, F.J., and Parkhurst, S.M. (1995). Signal transduction by cAMPdependent protein kinase A in Drosophila limb patterning. Nature 373, 711–715. Leshko-Lindsay, L. and Corces, V.G. (1997). The role of selectins in Drosophila eye and bristle development. Development 124, 169–180. Lesokhin, A.M., Yu, S.-Y., Katz, J., and Baker, N.E. (1999). Several levels of EGR receptor signaling during photoreceptor speciﬁcation in wild-type, Ellipse, and null mutant Drosophila. Dev. Biol. 205, 129–144. Lessing, D. and Nusse, R. (1998). Expression of wingless in the Drosophila embryo: a conserved cis-acting element lacking conserved Ci-binding sites is required for patchedmediated repression. Development 125, 1469–1476. Letsou, A., Arora, K., Wrana, J.L., Simin, K., Twombly, V.,

REFERENCES

2496.

2497.

2498. 2499.

2500.

2501. 2502.

2503.

2504. 2505. 2506. 2507. 2508.

2509. 2510. 2511.

2512.

2513.

2514. 2515.

Jamal, J., Staehling-Hampton, K., Hoffmann, F.M., Gelbart, W.M., Massagu´e, J., and O’Connor, M.B. (1995). Drosophila Dpp signaling is mediated by the punt gene product: a dual ligand-binding type II receptor of the TGFβ receptor family. Cell 80, 899–908. Levanon, D., Goldstein, R.E., Bernstein, Y., Tang, H., Goldenberg, D., Stifani, S., Paroush, Z., and Groner, Y. (1998). Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors. Proc. Natl. Acad. Sci. USA 95, 11590–11595. Levine, A., Weiss, C., and Wides, R. (1997). Expression of the pair-rule gene odd Oz (odz) in imaginal tissues. Dev. Dynamics 209, 1–14. Levine, M. and Hoey, T. (1988). Homeobox proteins as sequence-speciﬁc transcription factors. Cell 55, 537–540. Leviten, M.W., Lai, E.C., and Posakony, J.W. (1997). The Drosophila gene Bearded encodes a novel small protein and shares 3 UTR sequence motifs with multiple Enhancer of split Complex genes. Development 124, 4039–4051. Leviten, M.W. and Posakony, J.W. (1996). Gain-of-function alleles of Bearded interfere with alternative cell fate decisions in Drosophila adult sensory organ development. Dev. Biol. 176, 264–283. Lewin, B. (1998). The mystique of epigenetics. Cell 93, 301–303. Lewis, C.T. (1970). Structure and function in some external receptors. In “Insect Ultrastructure,” 5th Symp. Roy. Entomol. Soc. Lond., (A.C. Neville, ed.). Blackwell: Oxford, pp. 59–76. Lewis, D.L., DeCamillis, M., and Bennett, R.L. (2000). Distinct roles of the homeotic genes Ubx and abd-A in beetle embryonic abdominal appendage development. Proc. Natl. Acad. Sci. USA 97, 4504–4509. Lewis, E. (1995). Remembering Sturtevant. Genetics 141, 1227–1230. Lewis, E.B. (1951). Pseudoallelism and gene evolution. Cold Spr. Harb. Symp. Quant. Biol. 16, 159–174. Lewis, E.B. (1963). Genes and developmental pathways. Amer. Zool. 3, 33–56. Lewis, E.B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276, 565–570. Lewis, E.B. (1992). Clusters of master control genes regulate the development of higher organisms. JAMA 267, 1524–1531. Lewis, E.B. (1994). Homeosis: the ﬁrst 100 years. Trends Genet. 10, 341–343. Lewis, E.B. (1998). The bithorax complex: the ﬁrst ﬁfty years. Int. J. Dev. Biol. 42, 403–415. Lewis, E.B., Knafels, J.D., Mathog, D.R., and Celniker, S.E. (1995). Sequence analysis of the cis-regulatory regions of the bithorax complex of Drosophila. Proc. Natl. Acad. Sci. USA 92, 8403–8407. Lewis, J. (1981). Simpler rules for epimorphic regeneration: the polar-coordinate model without polar coordinates. J. Theor. Biol. 88, 371–392. Lewis, J. (1982). Continuity and discontinuity in pattern formation. In “Developmental Order: Its Origin and Regulation,” Symp. Soc. Dev. Biol., Vol. 40 (S. Subtelny and P.B. Green, eds.). Liss: New York, pp. 511–531. Lewis, J. (1998). A short cut to the nucleus. Nature 393, 304–305. Lewis, J., Slack, J.M.W., and Wolpert, L. (1977). Thresholds in development. J. Theor. Biol. 65, 579–590.

375

2516. Lewis, J.H. and Wolpert, L. (1976). The principle of nonequivalence in development. J. Theor. Biol. 62, 479–490. 2517. Lewis, P.H. (1947). Pc: Polycomb. Dros. Info. Serv. 21, 69. 2518. Lewontin, R. (2000). Computing the organism. Nat. Hist. 109 #3, 94. 2519. Leyns, L., Dambly-Chaudi`ere, C., and Ghysen, A. (1989). Two different sets of cis elements regulate scute to establish two different sensory patterns. Roux s Arch. Dev. Biol. 198, 227–232. 2520. Leyns, L., G´omez-Skarmeta, J.-L., and Dambly-Chaudi`ere, C. (1996). iroquois: a prepattern gene that controls the formation of bristles on the thorax of Drosophila. Mechs. Dev. 59, 63–72. 2521. Li, E. (1999). The mojo of methylation. Nature Genet. 23, 5–6. 2522. Li, L. and Vaessin, H. (2000). Pan-neural Prospero terminates cell proliferation during Drosophila neurogenesis. Genes Dev. 14, 147–151. 2523. Li, L., Yuan, H., Weaver, C.D., Mao, J., Farr III, G.H., Sussman, D.J., Jonkers, J., Kimelman, D., and Wu, D. (1999). Axin and Frat1 interact with Dv 1 and GSK, bridging Dv 1 to GSK in Wnt-mediated regulation of LEF-1. EMBO J. 18, 4233–4240. 2524. Li, L.-H. and Gergen, J.P. (1999). Differential interactions between Brother proteins and Runt domain proteins in the Drosophila embryo and eye. Development 126, 3313– 3322. [See also 2001 Development 128, 2639–2648.] 2525. Li, P., He, X., Gerrero, M.R., Mok, M., Aggarwal, A., and Rosenfeld, M.G. (1993). Spacing and orientation of bipartite DNA-binding motifs as potential functional determinants for POU domain factors. Genes Dev. 7, 2483–2496. 2526. Li, P., Yang, X., Wasser, M., Cai, Y., and Chia, W. (1997). Inscuteable and Staufen mediate asymmetric localization and segregation of prospero RNA during Drosophila neuroblast cell divisions. Cell 90, 437–447. 2527. Li, Q.-J., Pazdera, T.M., and Minden, J.S. (1999). Drosophila embryonic pattern repair: how embryos respond to cyclin E-induced ectopic division. Development 126, 2299–2307. 2528. Li, S., Auﬁero, B., Schiltz, R.L., and Walsh, M.J. (2000). Regulation of the homeodomain CCAAT displacement/cut protein function by histone acetyltransferases p300/CREB-binding protein (CBP)-associated factor and CBP. Proc. Natl. Acad. Sci. USA 97, 7166–7171. 2529. Li, S., Li, Y., Carthew, R.W., and Lai, Z.-C. (1997). Photoreceptor cell differentiation requires regulated proteolysis of the transcriptional repressor Tramtrack. Cell 90, 469–478. 2530. Li, S.-C., Songyang, Z., Vincent, S.J.F., Zwahlen, C., Wiley, S., Cantley, L., Kay, L.E., Forman-Kay, J., and Pawson, T. (1997). High-afﬁnity binding of the Drosophila Numb phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif. Proc. Natl. Acad. Sci. USA 94, 7204– 7209. 2531. Li, W., Melnick, M., and Perrimon, N. (1998). Dual function of Ras in Raf activation. Development 125, 4999–5008. 2532. Li, W., Noll, E., and Perrimon, N. (2000). Identiﬁcation of autosomal regions involved in Drosophila Raf function. Genetics 156, 763–774. 2533. Li, W., Ohlmeyer, J.T., Lane, M.E., and Kalderon, D. (1995). Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development. Cell 80, 553–562. 2534. Li, X., Gutjahr, T., and Noll, M. (1993). Separable regulatory elements mediate the establishment and maintenance of

376

2535.

2536.

2537.

2538.

2539.

2540.

2541.

2542.

2543.

2544.

2545.

2546.

2547. 2548.

2549.

2550.

REFERENCES

cell states by the Drosophila segment-polarity gene gooseberry. EMBO J. 12, 1427–1436. Li, X., Murre, C., and McGinnis, W. (1999). Activity regulation of a Hox protein and a role for the homeodomain in inhibiting transcriptional activation. EMBO J. 18, 198–211. Li, X. and Noll, M. (1993). Role of the gooseberry gene in Drosophila embryos: maintenance of wingless expression by a wingless-gooseberry autoregulatory loop. EMBO J. 12, 4499–4509. Li, X. and Noll, M. (1994). Compatibility between enhancers and promoters determines the transcriptional speciﬁcity of gooseberry and gooseberry neuro in the Drosophila embryo. EMBO J. 13, 400–406. Li, X. and Noll, M. (1994). Evolution of distinct developmental functions of three Drosophila genes by acquisition of different cis-regulatory regions. Nature 367, 83–87. Li, X., Veraksa, A., and McGinnis, W. (1999). A sequence motif distinct from Hox binding sites controls the speciﬁcity of a Hox response element. Development 126, 5581– 5589. Li, Y. and Baker, N.E. (2001). Proneural enhancement by Notch overcomes Suppressor-of-Hairless repressor function in the developing Drosophila eye. Curr. Biol. 11, 330– 338. Liang, Z. and Biggin, M.D. (1998). Eve and ftz regulate a wide array of genes in blastoderm embryos: the selector homeoproteins directly or indirectly regulate most genes in Drosophila. Development 125, 4471–4482. Lieber, T., Kidd, S., Alcamo, E., Corbin, V., and Young, M.W. (1993). Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei. Genes Dev. 7, 1949–1965. Lieber, T., Wesley, C.S., Alcamo, E., Hassel, B., Krane, J.F., Campos-Ortega, J.A., and Young, M.W. (1992). Single amino acid substitutions in EGF-like elements of Notch and Delta modify Drosophila development and affect cell adhesion in vitro. Neuron 9, 847–859. Lienhard, M.C. and Stocker, R.F. (1987). Sensory projection patterns of supernumerary legs and aristae in D. melanogaster. J. Exp. Zool. 244, 187–201. Lienhard, M.C. and Stocker, R.F. (1991). The development of the sensory neuron pattern in the antennal disc of wildtype and mutant (lz 3 , ss a ) Drosophila melanogaster. Development 112, 1063–1075. Lietzke, S.E., Bose, S., Cronin, T., Klarlund, J., Chawla, A., Czech, M.P., and Lambright, D.G. (2000). Structural basis of 3-phosphoinositide recognition by pleckstrin homology domains. Molec. Cell 6, 385–394. Lifson, S. (1994). What is information for molecular biology? BioEssays 16, 373–375. Ligoxygakis, P., Bray, S.J., Apidianakis, Y., and Delidakis, C. (1999). Ectopic expression of individual E(spl) genes has differential effects on different cell fate decisions and underscores the biphasic requirement for Notch activity in wing margin establishment in Drosophila. Development 126, 2205–2214. Ligoxygakis, P., Yu, S.-Y., Delidakis, C., and Baker, N.E. (1998). A subset of Notch functions during Drosophila eye development require Su(H) and the E(spl) gene complex. Development 125, 2893–2900. Lill, N.L., Douillard, P., Awwad, R.A., Ota, S., Lupher, M.L., Jr., Miyake, S., Meissner-Lula, N., Hsu, V.W., and Band, H. (2000). The evolutionarily conserved N-terminal region of

2551.

2552.

2553.

2554.

2555.

2556.

2557.

2558.

2559.

2560.

2561. 2562.

2563.

2564.

2565. 2566.

2567.

2568.

2569.

Cbl is sufﬁcient to enhance down-regulation of the epidermal growth factor receptor. J. Biol. Chem. 275#1, 367–377. Lilly, M.A. and Spradling, A.C. (1996). The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion. Genes Dev. 10, 2514–2526. Lim, W.A., Richards, F.M., and Fox, R.O. (1994). Structural determinants of peptide-binding orientation and of sequence speciﬁcity in SH3 domains. Nature 372, 375–379. Lin, D.M., Fetter, R.D., Kopczynski, C., Grenningloh, G., and Goodman, C.S. (1994). Genetic analysis of Fasciclin II in Drosophila: defasciculation, refasciculation, and altered fasciculation. Neuron 13, 1055–1069. Lin, H., Cho, S., and Cadigan, K.M. (2001). Split ends, a putative tissue-speciﬁc factor in Wingless signaling. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a163. Lin, X., Buff, E.M., Perrimon, N., and Michelson, A.M. (1999). Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development. Development 126, 3715–3723. Lin, X. and Perrimon, N. (1999). Dally cooperates with Drosophila Frizzled 2 to transduce Wingless signalling. Nature 400, 281–284. Lindenmayer, A. (1975). Developmental algorithms for multicellular organisms: a survey of L-systems. J. Theor. Biol. 54, 3–22. Lindenmayer, A. (1982). Developmental algorithms: lineage versus interactive control mechanisms. In “Developmental Order: Its Origin and Regulation,” Symp. Soc. Dev. Biol., Vol. 40 (S. Subtelny and P.B. Green, eds.). Alan R. Liss: New York, pp. 219–245. Lindenmayer, A. and Prusinkiewicz, P. (1989). Developmental models of multicellular organisms: a computer graphics perspective. In “Artiﬁcial Life,” (C.G. Langton, ed.). Addison-Wesley: New York, pp. 221–249. Lindsley, D.L. and Grell, E.H. (1968). “Genetic Variations of Drosophila melanogaster.” Carnegie Inst. Wash. Publ., Vol. 627. Washington, D. C. Lindsley, D.L. and Zimm, G.G. (1992). “The Genome of Drosophila melanogaster.” Acad. Pr., New York. Lipshitz, H.D. and Kankel, D.R. (1985). Developmental interactions between the peripheral and central nervous system in Drosophila melanogaster: analysis of the mutant, two-faced. Dev. Biol. 107, 1–12. Lisbin, M.J., Gordon, M., Yannoni, Y.M., and White, K. (2000). Function of RRM domains of Drosophila melanogaster ELAV: RNP1 mutations and RRM domain replacements with ELAV family proteins and SXL. Genetics 155, 1789–1798. Little, J.W., Byrd, C.A., and Brower, D.L. (1990). Effect of abx, bx and pbx mutations on expression of homeotic genes in Drosophila larvae. Genetics 124, 899–908. Little, J.W., Shepley, D.P., and Wert, D.W. (1999). Robustness of a gene regulatory circuit. EMBO J. 18, 4299–4307. Littlewood, T.D. and Evan, G.I. (1998). “Helix-loop-helix Transcription Factors.” 3rd ed. Protein Proﬁle Series, Oxford Univ. Pr., Oxford. Liu, D., Bienkowska, J., Petosa, C., Collier, R.J., Fu, H., and Liddington, R. (1995). Crystal structure of the zeta isoform of the 14-3-3 protein. Nature 376, 191–194. Liu, F., Hata, A., Baker, J.C., Doody, J., C´arcamo, J., Harland, R.M., and Massagu´e, J. (1996). A human Mad protein acting as a BMP-regulated transcriptional activator. Nature 381, 620–623. Liu, H., Ma, C., and Moses, K. (1996). Identiﬁcation

REFERENCES

2570.

2571.

2572.

2573.

2574.

2575.

2576.

2577. 2578.

2579.

2580.

2581.

2582.

2583.

2584.

2585. 2586. 2587.

and functional characterization of conserved promoter elements from glass : a retinal development gene of Drosophila. Mechs. Dev. 56, 73–82. Liu, X., Grammont, M., and Irvine, K.D. (2000). Roles for scalloped and vestigial in regulating cell afﬁnity and interactions between the wing blade and the wing hinge. Dev. Biol. 228, 287–303. Liu, X. and Lengyel, J.A. (2000). Drosophila arc encodes a novel adherens junction-associated PDZ domain protein required for wing and eye development. Dev. Biol. 221, 419–434. Liu, Z.-P., Galindo, R.L., and Wasserman, S.A. (1997). A role for CKII phosphorylation of the Cactus PEST domain in dorsoventral patterning of the Drosophila embryo. Genes Dev. 11, 3413–3422. Livneh, E., Glazer, L., Segal, D., Schlessinger, J., and Shilo, B.-Z. (1985). The Drosophila EGF receptor gene homolog: conservation of both hormone binding and kinase domains. Cell 40, 599–607. Llimargas, M. (1999). The Notch pathway helps to pattern the tips of the Drosophila tracheal branches by selecting cell fates. Development 126, 2355–2364. Llimargas, M. and Casanova, J. (1997). ventral veinless, a POU domain transcription factor, regulates different transduction pathways required for tracheal branching in Drosophila. Development 124, 3273–3281. Llimargas, M. and Casanova, J. (1999). EGF signalling regulates cell invagination as well as cell migration during formation of tracheal system in Drosophila. Dev. Genes Evol. 209, 174–179. Lloyd, C.W. and Rees, D.A., eds. (1981). “Cellular Controls in Differentiation.” Acad. Pr., New York. Lo, P.C.H. and Frasch, M. (1999). Sequence and expression of myoglianin, a novel Drosophila gene of the TGF-β superfamily. Mechs. Dev. 86, 171–175. Lo, R.S., Chen, Y.-G., Shi, Y., Pavletich, N.P., and Massagu´e, J. (1998). The L3 loop: a structural motif determining speciﬁc interactions between SMAD proteins and TGF-β receptors. EMBO J. 17, 996–1005. Lo, S.H., Weisberg, E., and Chen, L.B. (1994). Tensin: a potential link between the cytoskeleton and signal transduction. BioEssays 16, 817–823. Locke, J. and Hanna, S. (1996). engrailed gene dosage determines whether certain recessive cubitus interruptus alleles exhibit dominance of the adult wing phenotype in Drosophila. Dev. Genet. 19, 340–349. Locke, J., Kotarski, M.A., and Tartof, K.D. (1988). Dosagedependent modiﬁers of position effect variegation in Drosophila and a mass action model that explains their effect. Genetics 120, 181–198. Locke, J., Podemski, L., and Ferrer, C. (2001). Inversion of the whole arm of chromosome 4 in Drosophila species. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a103. [See also 2001 Chromosoma 110, 305–312.] Locke, M. (1984). Epidermal cells [Arthropoda]. In “Biology of the Integument,” Vol. 1 (J. Bereiter-Hahn, A.G. Matoltsy, and K.S. Richards, eds.). Springer-Verlag: Berlin, pp. 502–522. Locke, M. (1985). The structure of epidermal feet during their development. Tissue & Cell 17, 901–921. Locke, M. (1987). The very rapid induction of ﬁlopodia in insect cells. Tissue & Cell 19, 301–318. Locke, M. and Huie, P. (1981). Epidermal feet in insect morphogenesis. Nature 293, 733–735.

377

2588. Locke, M. and Huie, P. (1981). Epidermal feet in pupal segment morphogenesis. Tissue & Cell 13, 787–803. 2589. Logeat, F., Bessia, C., Brou, C., LeBail, O., Jarriault, S., Seidah, N.G., and Isra¨el, A. (1998). The Notch1 receptor is cleaved constitutively by a furin-like convertase. Proc. Natl. Acad. Sci. USA 95, 8108–8112. 2590. L¨ohr, U., Yussa, M., and Pick, L. (2001). Drosophila fushi tarazu: a gene on the border of homeotic function. Curr. Biol. 11, 1403–1412. [See also 2001 Curr. Biol. 11, 1473– 1478.] 2591. Lohs-Schardin, M., Sander, K., Cremer, C., Cremer, T., and Zorn, C. (1979). Localized ultraviolet laser microbeam irradiation of early Drosophila embryos: fate maps based on location and frequency of adult defects. Dev. Biol. 68, 533–545. 2592. Long, A.D., Lyman, R.F., Langley, C.H., and Mackay, T.F.C. (1998). Two sites in the Delta gene region contribute to naturally occurring variation in bristle number in Drosophila melanogaster. Genetics 149, 999–1017. 2593. Long, A.D., Lyman, R.F., Morgan, A.H., Langley, C.H., and Mackay, T.F.C. (2000). Both naturally occurring insertions of transposable elements and intermediate frequency polymorphisms at the achaete-scute complex are associated with variation in bristle number in Drosophila melanogaster. Genetics 154, 1255–1269. 2594. Longley, R.L., Jr. and Ready, D.F. (1995). Integrins and the development of three-dimensional structure in the Drosophila compound eye. Dev. Biol. 171, 415–433. 2595. Lonie, A., D’Andrea, R., Paro, R., and Saint, R. (1994). Molecular characterization of the Polycomblike gene of Drosophila melanogaster, a trans-acting negative regulator of homeotic gene expression. Development 120, 2629– 2636. 2596. Loo, L.W., Yost, C., and Eisenman, R.N. (2001). Functional characterization of dMad, a transcriptional repressor, during development. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a125. 2597. Loomis, W.F. and Kuspa, A. (1992). Spontaneous generation of enhancers by point mutations. Trends Genet. 8, 229. 2598. Lopez, A., Salvaing, J., Deutsch, J., and Peronnet, F. (2001). Involvement of CORTO in Polycomb and trithorax functions. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a96. 2599. L´opez, A.J. (1995). Developmental role of transcription factor isoforms generated by alternative splicing. Dev. Biol. 172, 396–411. 2600. Lopez, A.J. and Hogness, D.S. (1991). Immunochemical dissection of the Ultrabithorax homeoprotein family in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 88, 9924–9928. 2601. Lopez, M., Oettgen, P., Akbarali, Y., Dendorfer, U., and Libermann, T.A. (1994). ERP, a new member of the ets transcription factor/oncoprotein family: cloning, characterization, and differential expression during B-lymphocyte development. Mol. Cell. Biol. 14, 3292–3309. 2602. Lopez-Schier, H. and St. Johnston, D. (2001). agoraphobic modulates Notch receptor cleavage and signalling in Drosophila. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a158. [Cf. 2002 Dev. Cell 2, 79–89.] 2603. Lorick, K.L., Jensen, J.P., Fang, S., Ong, A.M., Hatakeyama, S., and Weissman, A.M. (1999). RING ﬁngers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc. Natl. Acad. Sci. USA 96, 11364–11369.

378

2604. Loureiro, J. and Peifer, M. (1998). Roles of Armadillo, a Drosophila catenin, during central nervous system development. Curr. Biol. 8, 622–632. 2605. Love, J.J., Li, X., Case, D.A., Giese, K., Grosschedl, R., and Wright, P.E. (1995). Structural basis for DNA bending by the architectural transcription factor LEF-1. Nature 376, 791–795. 2606. Lu, B., Ackerman, L., Jan, L.Y., and Jan, Y.-N. (1999). Modes of protein movement that lead to the asymmetric localization of Partner of Numb during Drosophila neuroblast division. Molec. Cell 4, 883–891. 2607. Lu, B., Jan, L.Y., and Jan, Y.-N. (1998). Asymmetric cell division: lessons from ﬂies and worms. Curr. Opin. Genet. Dev. 8, 392–399. 2608. Lu, B., Roegiers, F., Jan, L.Y., and Jan, Y.N. (2001). Adherens junctions inhibit asymmetric division in the Drosophila epithelium. Nature 409, 522–525. 2609. Lu, B., Rothenberg, M., Jan, L.Y., and Jan, Y.N. (1998). Partner of Numb colocalizes with Numb during mitosis and directs Numb asymmetric localization in Drosophila neural and muscle progenitors. Cell 95, 225–235. 2610. Lu, C.-H., Rinc´on-Limas, D.E., and Botas, J. (2000). Conserved overlapping and reciprocal expression of msh/Msx1 and apterous/Lhx2 in Drosophila and mice. Mechs. Dev. 99, 177–181. 2611. Lu, F.M. and Lux, S.E. (1996). Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element. Proc. Natl. Acad. Sci. USA 93, 5663–5667. 2612. Lu, X. and Li, Y. (1999). Drosophila Src42A is a negative regulator of RTK signaling. Dev. Biol. 208, 233–243. 2613. Lu, X., Melnick, M.B., Hsu, J.-C., and Perrimon, N. (1994). Genetic and molecular analyses of mutations involved in Drosophila raf signal transduction. EMBO J. 13, 2592– 2599. 2614. Lucchesi, J.C. (1983). Curt Stern: 1902–1981. Genetics 103, 1–4. 2615. Lucchesi, J.C. (1994). Sturtevant’s mantle and the (lost?) art of chromosome mechanics. Genetics 136, 707–708. 2616. Ludwig, M.Z., Patel, N.H., and Kreitman, M. (1998). Functional analysis of eve stripe 2 enhancer evolution in Drosophila: rules governing conservation and change. Development 125, 949–958. 2617. Lunde, K., Biehs, B., Nauber, U., and Bier, E. (1998). The knirps and knirps-related genes organize development of the second wing vein in Drosophila. Development 125, 4145–4154. 2618. Luo, H., Asha, H., Kockel, L., Parke, T., Mlodzik, M., and Dearolf, C.R. (1999). The Drosophila Jak kinase Hopscotch is required for multiple developmental processes in the eye. Dev. Biol. 213, 432–441. [See also 2001 BioEssays 23, 1138–1147.] 2619. Luo, K. and Lodish, H.F. (1997). Positive and negative regulation of type II TGF-β receptor signal transduction by autophosphorylation on multiple serine residues. EMBO J. 16, 1970–1981. 2620. Luo, Z., Tzivion, G., Belshaw, P.J., Vavvas, D., Marshall, M., and Avruch, J. (1996). Oligomerization activates cRaf-1 through a Ras-dependent mechanism. Nature 383, 181–185. 2621. Lupas, A. (1996). Coiled coils: new structures and new functions. Trends Biochem. Sci. 21, 375–382. 2622. Lupo, R., Breiling, A., Bianchi, M.E., and Orlando, V. (2001).

REFERENCES

2623.

2624.

2625. 2626.

2627.

2628.

2629.

2630.

2631.

2632.

2633.

2634.

2635.

2636.

2637. 2638.

2639.

Drosophila chromosome condensation proteins Topoisomerase II and Barren colocalize with Polycomb and maintain Fab-7 PRE silencing. Molec. Cell 7, 127–136. Luschnig, S., Krauss, J., Bohmann, K., Desjeux, I., and ¨ Nusslein-Volhard, C. (2000). The Drosophila SHC adaptor protein is required for signaling by a subset of receptor tyrosine kinases. Molec. Cell 5, 231–241. Lux, S.E., John, K.M., and Bennett, V. (1990). Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cellcycle control proteins. Nature 344, 36–42. Lyko, F. (2001). DNA methylation learns to ﬂy. Trends Genet. 17, 169–172. Lyman, D. and Young, M.W. (1993). Further evidence for function of the Drosophila Notch protein as a transmembrane receptor. Proc. Natl. Acad. Sci. USA 90, 10395–10399. Lyman, D.F. and Yedvobnick, B. (1995). Drosophila Notch receptor activity suppresses Hairless function during adult external sensory organ development. Genetics 141, 1491– 1505. Lyman, R.F. and Mackay, T.F.C. (1998). Candidate quantitative trait loci and naturally occurring phenotypic variation for bristle number in Drosophila melanogaster : the Delta-Hairless gene region. Genetics 149, 983–998. Lynch, M. and Conery, J.S. (2000). The evolutionary fate and consequences of duplicate genes. Science 290, 1151– 1155. Lynch, M. and Force, A. (2000). The probability of duplicate gene preservation by subfunctionalization. Genetics 154, 459–473. Ma, C. and Moses, K. (1995). wingless and patched are negative regulators of the morphogenetic furrow and can affect tissue polarity in the developing Drosophila compound eye. Development 121, 2279–2289. Ma, C., Zhou, Y., Beachy, P.A., and Moses, K. (1993). The segment polarity gene hedgehog is required for progression of the morphogenetic furrow in the developing Drosophila eye. Cell 75, 927–938. Ma, J. and Karplus, M. (1997). Molecular switch in signal transduction: reaction paths of the conformational changes in ras p21. Proc. Natl. Acad. Sci. USA 94, 11905– 11910. Ma, P.C.M., Rould, M.A., Weintraub, H., and Pabo, C.O. (1994). Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation. Cell 77, 451– 459. Ma, Y., Niemitz, E.L., Nambu, P.A., Shan, X., Sackerson, C., Fujioka, M., Goto, T., and Nambu, J.R. (1998). Gene regulatory functions of Drosophila Fish-hook, a high mobility group domain Sox protein. Mechs. Dev. 73, 169–182. ¨ Maas, A.-H. (1948). Uber die Ausl¨osbarkeit von Temperatur-Modiﬁkationen w¨ahrend der EmbryonalEntwicklung von Drosophila melanogaster Meigen. W. Roux’ Arch. Entw.-Mech. Org. 143, 515–572. Macara, I.G. (1999). Nuclear transport: randy couples. Curr. Biol. 9, R436–R439. MacBean, I.T., McKenzie, J.A., and Parsons, P.A. (1971). A pair of closely linked genes controlling high scutellar chaeta number in Drosophila. Theor. Appl. Genet. 41, 227– 235. MacDougall, L.K. and Waterﬁeld, M.D. (1996). Receptor signalling: to Sevenless, a daughter. Curr. Biol. 6, 1250– 1253.

REFERENCES

2640. MacDowell, E.C. (1915). Bristle inheritance in Drosophila. I. Extra bristles. J. Exp. Zool. 19, 61–97. 2641. Mac´ıas, A. and Morata, G. (1996). Functional hierarchy and phenotypic suppression among Drosophila homeotic genes: the labial and empty spiracles genes. EMBO J. 15, 334–343. 2642. Mac´ıas, A., Pelaz, S., and Morata, G. (1994). Genetic factors controlling the expression of the abdominal-A gene of Drosophila within its domain. Mechs. Dev. 46, 15–25. 2643. MacKay, T.F.C. (1995). The genetic basis of quantitative variation: numbers of sensory bristles of Drosophila melanogaster as a model system. Trends Genet. 11, 464– 470. 2644. Mackay, T.F.C. (1995). The nature of quantitative genetic variation revisited: lessons from Drosophila bristles. BioEssays 18, 113–121. 2645. Mackay, T.F.C. and Langley, C.H. (1990). Molecular and phenotypic variation in the achaete-scute region of Drosophila melanogaster. Nature 348, 64–65. 2646. MacWilliams, H.K. (1978). A model of gradient interpretation based on morphogen binding. J. Theor. Biol. 72, 385–411. 2647. Madhani, H.D. and Fink, G.R. (1998). The riddle of MAP kinase signaling speciﬁcity. Trends Genet. 14, 151–155. 2648. Madhavan, M.M. and Madhavan, K. (1980). Morphogenesis of the epidermis of adult abdomen of Drosophila. J. Embryol. Exp. Morph. 60, 1–31. 2649. Madhavan, M.M. and Madhavan, K. (1982). Pattern regulation in tergite of Drosophila: a model. J. Theor. Biol. 95, 731–748. 2650. Madhavan, M.M. and Schneiderman, H.A. (1977). Histological analysis of the dynamics of growth of imaginal discs and histoblast nests during the larval development of Drosophila melanogaster. W. Roux’s Arch. 183, 269– 305. 2651. Madore, B.F. and Freedman, W.L. (1987). Self-organizing structures. Am. Sci. 75, 252–259. 2652. Maegley, K.A., Admiraal, S.J., and Herschlag, D. (1996). Ras-catalyzed hydrolysis of GTP: a new perspective from model studies. Proc. Natl. Acad. Sci. USA 93, 8160–8166. 2653. Magee, T. and Marshall, C. (1999). New insights into the interaction of Ras with the plasma membrane. Cell 98, 9–12. 2654. Mahadevan, L.C. (1991). Growth factors, intracellular signals and developmental decisions: can growth factors be morphogens? Sems. Dev. Biol. 2, 339–343. 2655. Mahaffey, J.W. and Kaufman, T.C. (1987). Distribution of Sex combs reduced gene products in Drosophila melanogaster. Genetics 117, 51–60. 2656. Mahaffey, J.W. and Kaufman, T.C. (1987). The homeotic genes of the Antennapedia Complex and the Bithorax Complex of Drosophila. In “Developmental Genetics of Higher Organisms,” (G.M. Malacinski, ed.). Macmillan: New York, pp. 329–359. 2657. Maier, D., Marquart, J., Thompson-Fontaine, A., Beck, I., Wurmbach, E., and Preiss, A. (1997). In vivo structurefunction analysis of Drosophila HAIRLESS. Mechs. Dev. 67, 97–106. 2658. Maier, D., Nagel, A.C., Johannes, B., and Preiss, A. (1999). Subcellular localization of Hairless protein shows a major focus of activity within the nucleus. Mechs. Dev. 89, 195– 199. 2659. Maier, D., Stumm, G., Kuhn, K., and Preiss, A. (1992). Hairless, a Drosophila gene involved in neural development,

379

2660.

2661.

2662.

2663.

2664.

2665.

2666.

2667.

2668.

2669.

2670.

2671. 2672. 2673.

2674. 2675.

2676.

2677.

encodes a novel, serine rich protein. Mechs. Dev. 38, 143– 156. Maixner, A., Hecker, T.P., Phan, Q.N., and Wassarman, D.A. (1998). A screen for mutations that prevent lethality caused by expression of activated Sevenless and Ras1 in the Drosophila embryo. Dev. Genet. 23, 347–361. Maizel, A., Bensaude, O., Prochiantz, A., and Joliot, A. (1999). A short region of its homeodomain is necessary for Engrailed nuclear export and secretion. Development 126, 3183–3190. Majumdar, A., Nagaraj, R., and Banerjee, U. (1997). strawberry notch encodes a conserved nuclear protein that functions downstream of Notch and regulates gene expression along the developing wing margin of Drosophila. Genes Dev. 11, 1341–1353. Makkerh, J.P.S., Dingwall, C., and Laskey, R.A. (1996). Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids. Curr. Biol. 6, 1025–1027. Maldonado-Codina, G. and Glover, D.M. (1992). Cyclins A and B associate with chromatin and the polar regions of spindles, respectively, and do not undergo complete degradation at anaphase in syncytial Drosophila embryos. J. Cell Biol. 116, 967–976. Malik, S. and Roeder, R.G. (2000). Transcriptional regulation through Mediator-like coactivators in yeast and metazoan cells. Trends Biochem. Sci. 25, 277–283. [See also 2001 references: Molec. Cell 8, 9–19; Development 128, 3095– 3104.] Mallin, D.R., Myung, J.S., Patton, J.S., and Geyer, P.K. (1998). Polycomb group repression is blocked by the Drosophila suppressor of Hairy-wing [su(Hw)] insulator. Genetics 148, 331–339. Manfruelli, P., Arquier, N., Hanratty, W.P., and S´em´eriva, M. (1996). The tumor suppressor gene, lethal(2)giant larvae (l(2)gl), is required for cell shape change of epithelial cells during Drosophila development. Development 122, 2283– 2294. Maniatis, T. (1999). A ubiquitin ligase complex essential for the NF-κB, Wnt/Wingless, and Hedgehog signaling pathways. Genes Dev. 13, 505–510. Mankidy, R., Abbeyquaye, T., and Thackeray, J.R. (2001). Genetic and molecular analysis of PLC-γ function. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a170. Mann, R.S. (1994). engrailed-mediated repression of Ultrabithorax is necessary for the parasegment 6 identity in Drosophila. Development 120, 3205–3212. Mann, R.S. (1995). The speciﬁcity of homeotic gene function. BioEssays 17, 855–863. Mann, R.S. (1997). Why are Hox genes clustered? BioEssays 19, 661–664. Mann, R.S. and Abu-Shaar, M. (1996). Nuclear import of the homeodomain protein Extradenticle in response to Wg and Dpp signalling. Nature 383, 630–633. Mann, R.S. and Affolter, M. (1998). Hox proteins meet more partners. Curr. Opin. Gen. Dev. 8, 423–429. Mann, R.S. and Chan, S.-K. (1996). Extra speciﬁcity from extradenticle: the partnership between HOX and PBX/EXD homeodomain proteins. Trends Genet. 12, 258– 262. Mann, R.S. and Hogness, D.S. (1990). Functional dissection of Ultrabithorax proteins in D. melanogaster. Cell 60, 597–610. Mann, R.S. and Morata, G. (2000). The developmental and

380

2678.

2679.

2680.

2681.

2682.

2683.

2684.

2685.

2686. 2687.

2688. 2689.

2690.

2691.

2692. 2693. 2694. 2695.

2696.

REFERENCES

molecular biology of genes that subdivide the body of Drosophila. Annu. Rev. Cell Dev. Biol. 16, 243–271. Mannervik, M., Nibu, Y., Zhang, H., and Levine, M. (1999). Transcriptional coregulators in development. Science 284, 606–609. Manning, G. and Krasnow, M.A. (1993). Development of the Drosophila tracheal system. In “The Development of Drosophila melanogaster,” Vol. 1 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Lab. Pr.: Plainview, N. Y., pp. 609–685. Manning, L. and Doe, C.Q. (1999). Prospero distinguishes sibling cell fate without asymmetric localization in the Drosophila adult external sense organ lineage. Development 126, 2063–2071. Manoukian, A.S. and Krause, H.M. (1992). Concentrationdependent activities of the even-skipped protein in Drosophila embryos. Genes Dev. 6, 1740–1751. Manoukian, A.S. and Krause, H.M. (1993). Control of segmental asymmetry in Drosophila embryos. Development 118, 785–796. Manoukian, A.S., Yoffe, K.B., Wilder, E.L., and Perrimon, N. (1995). The porcupine gene is required for wingless autoregulation in Drosophila. Development 121, 4037– 4044. Manseau, L., Baradaran, A., Brower, D., Budhu, A., Elefant, F., Phan, H., Philp, A.V., Yang, M., Glover, D., Kaiser, K., Palter, K., and Selleck, S. (1997). GAL4 enhancer traps expressed in the embryo, larval brain, imaginal discs, and ovary of Drosophila. Dev. Dynamics 209, 310–322. Mansﬁeld, E., Hersperger, E., Biggs, J., and Shearn, A. (1994). Genetic and molecular analysis of hyperplastic discs, a gene whose product is required for regulation of cell proliferation in Drosophila melanogaster imaginal discs and germ cells. Dev. Biol. 165, 507–526. Maquat, L.E. and Carmichael, G.G. (2001). Quality control of mRNA function. Cell 104, 173–176. Marcu, O., Cros, N., and Marsh, J.L. (2001). Identiﬁcation of wg autoregulatory elements. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a115. Marcus, J.M. (2001). The development and evolution of crossveins in insect wings. J. Anat. 199, 211–216. Mardon, G., Solomon, N.M., and Rubin, G.M. (1994). dachshund encodes a nuclear protein required for normal eye and leg development in Drosophila. Development 120, 3473–3486. Mar´ı-Beffa, M., de Celis, J.F., and Garc´ıa-Bellido, A. (1991). Genetic and developmental analyses of chaetae pattern formation in Drosophila tergites. Roux’s Arch. Dev. Biol. 200, 132–142. Marigo, V., Davey, R.A., Zuo, Y., Cunningham, J.M., and Tabin, C.J. (1996). Biochemical evidence that Patched is the Hedgehog receptor. Nature 384, 176–179. Markert, C.L. and Ursprung, H. (1971). “Developmental Genetics.” Prentice-Hall, Englewood Cliffs, N. J. Markow, T.A., ed. (1994). “Developmental Instability: Its ¨ Origins and Evolutionary Implications.” Kluwer, London. Markus, M. and Hess, B. (1990). Isotropic cellular automaton for modelling excitable media. Nature 347, 56–58. Marquart, J., Alexief-Damianof, C., Preiss, A., and Maier, D. (1999). Rapid divergence in the course of Drosophila evolution reveals structural important domains of the Notch antagonist Hairless. Dev. Genes Evol. 209, 155–164. ¨ Marqu´es, G., Musacchio, M., Shimell, M.J., WunnenbergStapleton, K., Cho, K.W.Y., and O’Connor, M.B. (1997). Production of a DPP activity gradient in the early Drosophila

2697.

2698. 2699. 2700.

2701.

2702. 2703.

2704.

2705. 2706. 2707.

2708.

2709.

2710. 2711.

2712.

2713.

2714.

2715.

embryo through the opposing actions of the SOG and TLD proteins. Cell 91, 417–426. Marsh, J.L., Syed, A., Theisen, H., and Sanchez, S. (2001). A novel function for Arm and dTcf: negative regulation of gene expression in response to Wg/Wnt signaling. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a206. Marsh, J.L. and Theisen, H. (1999). Regeneration in insects. Sems. Cell Dev. Biol. 10, 365–375. Marsh, M. and McMahon, H.T. (1999). The structural era of endocytosis. Science 285, 215–220. Marshall, C.J. (1994). MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr. Opin. Gen. Dev. 4, 82–89. Marshall, C.J. (1995). Speciﬁcity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80, 179–185. Marshall, C.J. (1996). Raf gets it together. Nature 383, 127– 128. Martin, C.H., Mayeda, C.A., Davis, C.A., Ericsson, C.L., Knafels, J.D., Mathog, D.R., Celniker, S.E., Lewis, E.B., and Palazzolo, M.J. (1995). Complete sequence of the bithorax complex of Drosophila. Proc. Natl. Acad. Sci. USA 92, 8398–8402. Martin, E.C. and Adler, P.N. (1993). The Polycomb group gene Posterior Sex Combs encodes a chromosomal protein. Development 117, 641–655. Martin, G. (1996). Pass the butter . . . Science 274, 203–204. Martin, G.R. (1995). Why thumbs are up. Nature 374, 410–411. Martin, J.F., Hersperger, E., Simcox, A., and Shearn, A. (2000). minidiscs encodes a putative amino acid transporter subunit required non-autonomously for imaginal cell proliferation. Mechs. Dev. 92, 155–167. Martin, K.A., Poeck, B., Roth, H., Ebens, A.J., Ballard, L.C., and Zipursky, S.L. (1995). Mutations disrupting neuronal connectivity in the Drosophila visual system. Neuron 14, 229–240. Martin, P., Martin, A., and Shearn, A. (1977). Studies of l(3)c43hs1 a polyphasic, temperature-sensitive mutant of Drosophila melanogaster with a variety of imaginal disc defects. Dev. Biol. 55, 213–232. Martin, P.F. (1982). Direct determination of the growth rate of Drosophila imaginal discs. J. Exp. Zool. 222, 97–102. Mart´ın, V., Carrillo, G., Torroja, C., and Guerrero, I. (2001). The sterol-sensing domain of Patched protein seems to control Smoothened activity through Patched vesicular trafﬁcking. Curr. Biol. 11, 601–607. Mart´ın-Bermudo, M.D., Carmena, A., and Jim´enez, F. (1995). Neurogenic genes control gene expression at the transcriptional level in early neurogenesis and in mesectoderm speciﬁcation. Development 121, 219–224. Mart´ın-Blanco, E. (1998). Regulatory control of signal transduction during morphogenesis in Drosophila. Int. J. Dev. Biol. 42, 363–368. Mart´ın-Blanco, E., Pastor-Pareja, J.C., and Garc´ıa-Bellido, A. (2000). JNK and decapentaplegic signaling control adhesiveness and cytoskeletal dynamics during thorax closure in Drosophila. Proc. Natl. Acad. Sci. USA 97, 7888– 7893. Mart´ın-Blanco, E., Roch, F., Noll, E., Baonza, A., Duffy, J.B., and Perrimon, N. (1999). A temporal switch in DER signaling controls the speciﬁcation and differentiation of veins and interveins in the Drosophila wing. Development 126, 5739–5747.

REFERENCES

2716. Martinez Arias, A. (1989). A cellular basis for pattern formation in the insect epidermis. Trends Genet. 5, 262–267. [See also 2001 Trends Genet. 17, 574–579.] 2717. Martinez Arias, A. (1993). Development and patterning of the larval epidermis of Drosophila. In “The Development of Drosophila melanogaster,” Vol. 1 (M. Bate and A. Martinez-Arias, eds.). Cold Spring Harbor Laboratory Pr.: Plainview, N. Y., pp. 517–608. 2718. Martinez Arias, A. (1998). Interactions between Wingless and Notch during the assignation of cell fates in Drosophila. Int. J. Dev. Biol. 42, 325–333. 2719. Martinez Arias, A., Baker, N.E., and Ingham, P.W. (1988). Role of segment polarity genes in the deﬁnition and maintenance of cell states in the Drosophila embryo. Development 103, 157–170. 2720. Martinez Arias, A., Brown, A.M.C., and Brennan, K. (1999). Wnt signalling: pathway or network? Curr. Opin. Gen. Dev. 9, 447–454. 2721. Mart´ınez, C. and Modolell, J. (1991). Cross-regulatory interactions between the proneural achaete and scute genes of Drosophila. Science 251, 1485–1487. 2722. Mart´ınez, C., Modolell, J., and Garrell, J. (1993). Regulation of the proneural gene achaete by helix-loop-helix proteins. Mol. Cell. Biol. 13, 3514–3521. 2723. Martinez-Arias, A., Ingham, P.W., Scott, M.P., and Akam, M.E. (1987). The spatial and temporal deployment of Dfd and Scr transcripts throughout development of Drosophila. Development 100, 673–683. 2724. Martinez-Arias, A. and Lawrence, P.A. (1985). Parasegments and compartments in the Drosophila embryo. Nature 313, 639–642. 2725. Martinez-Arias, A. and White, R.A.H. (1988). Ultrabithorax and engrailed expression in Drosophila embryos mutant for segmentation genes of the pair-rule class. Development 102, 325–338. 2726. Mart´ınez-Balb´as, M.A., Bannister, A.J., Martin, K., HausSeuffert, P., Meisterernst, M., and Kouzarides, T. (1998). The acetyltransferase activity of CBP stimulates transcription. EMBO J. 17, 2886–2893. ¨ 2727. Marty, T., Muller, B., Basler, K., and Affolter, M. (2000). Schnurri mediates Dpp-dependent repression of brinker transcription. Nature Cell Biol. 2, 745–749. [See also 2001 EMBO J. 20, 3298–3305.] 2728. Maschat, F., Serrano, N., Randsholt, N.B., and G´eraud, G. (1998). engrailed and polyhomeotic interactions are required to maintain the A/P boundary of the Drosophila developing wing. Development 125, 2771–2780. 2729. Maschat, F., Toulza, F., and Montagne, J. (2001). Engrailed and D-SRF are acting as cofactors on polyhomeotic activation during wing morphogenesis. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a187. 2730. Mason, E.D., Williams, S., Grotendorst, G.R., and Marsh, J.L. (1997). Combinatorial signaling by Twisted Gastrulation and Decapentaplegic. Mechs. Dev. 64, 61– 75. 2731. Massagu´e, J. (1992). Receptors for the TGF-β family. Cell 69, 1067–1070. 2732. Massagu´e, J. (1996). TGFβ signaling: receptors, transducers, and Mad proteins. Cell 85, 947–950. 2733. Massagu´e, J. (1998). TGF-β signal transduction. Annu. Rev. Biochem. 67, 753–791. 2734. Massagu´e, J., Attisano, L., and Wrana, J.L. (1994). The TGFβ family and its composite receptors. Trends Cell Biol. 4, 172–178. 2735. Massagu´e, J., Blain, S.W., and Lo, R.S. (2000). TGFβ signal-

381

2736.

2737.

2738.

2739.

2740.

2741.

2742.

2743.

2744. 2745.

2746.

2747.

2748.

2749.

2750. 2751.

2752.

ing in growth control, cancer, and heritable disorders. Cell 103, 295–309. Massagu´e, J. and Wotton, D. (2000). Transcriptional control by the TGF-β/Smad signaling system. EMBO J. 19, 1745–1754. Massari, M.E., Grant, P.A., Pray-Grant, M.G., Berger, S.L., Workman, J.L., and Murre, C. (1999). A conserved motif present in a class of helix-loop-helix proteins activates transcription by direct recruitment of the SAGA complex. Molec. Cell 4, 63–73. Masucci, J.D. and Hoffmann, F.M. (1993). Identiﬁcation of two regions from the Drosophila decapentaplegic gene required for embryonic midgut development and larval viability. Dev. Biol. 159, 276–287. Masucci, J.D., Miltenberger, R.J., and Hoffmann, F.M. (1990). Pattern-speciﬁc expression of the Drosophila decapentaplegic gene in imaginal discs is regulated by 3 cisregulatory elements. Genes Dev. 4, 2011–2023. Mata, J., Curado, S., Ephrussi, A., and Rørth, P. (2000). Tribbles coordinates mitosis and morphogenesis in Drosophila by regulating String/CDC25 proteolysis. Cell 101, 511–522. Matakatsu, H., Brentrup, D., and Hayashi, S. (2001). Repression of wing vein differentiation by interaction between Plexus and two bHLH proteins. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a200. Matakatsu, H., Tadokoro, R., Gamo, S., and Hayashi, S. (1999). Repression of the wing vein development in Drosophila by the nuclear matrix protein Plexus. Development 126, 5207–5216. M´ath´e, E., Bates, H., Huikeshoven, H., De´ak, P., Glover, D.M., and Cotterill, S. (2000). Importin-α3 is required at multiple stages of Drosophila development and has a role in the completion of oogenesis. Dev. Biol. 223, 307–322. Mathi, S.K. and Larsen, E. (1988). Patterns of cell division in imaginal discs of Drosophila. Tissue & Cell 20, 461–472. Mathog, D.R. (1991). Suppression of abdominal legs in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 199, 449– 457. Matsuno, K., Diederich, R.J., Go, M.J., Blaumueller, C.M., and Artavanis-Tsakonas, S. (1995). Deltex acts as a positive regulator of Notch signaling through interactions with the Notch ankyrin repeats. Development 121, 2633–2644. Matsuno, K., Go, M.J., Sun, X., Eastman, D.S., and Artavanis-Tsakonas, S. (1997). Suppressor of Hairlessindependent events in Notch signaling imply novel pathway elements. Development 124, 4265–4273. Matsuo, T., Takahashi, K., Kondo, S., Kaibuchi, K., and Yamamoto, D. (1997). Regulation of cone cell formation by Canoe and Ras in the developing Drosophila eye. Development 124, 2671–2680. Matsuzaki, F., Ohshiro, T., Ikeshima-Kataoka, H., and Izumi, H. (1998). miranda localizes staufen and prospero asymmetrically in mitotic neuroblasts and epithelial cells in early Drosophila embryogenesis. Development 125, 4089–4098. Mattaj, I.W. and Conti, E. (1999). Snail mail to the nucleus. Nature 399, 208–210. Matunis, E., Tran, J., G¨onczy, P., Caldwell, K., and DiNardo, S. (1997). punt and schnurri regulate a somatically derived signal that restricts proliferation of committed progenitors in the germline. Development 124, 4383–4391. Maurel-Zaffran, C. and Treisman, J.E. (2000). pannier acts upstream of wingless to direct dorsal eye disc development in Drosophila. Development 127, 1007–1016.

382

2753. Maves, L. and Schubiger, G. (1995). wingless induces transdetermination in developing Drosophila imaginal discs. Development 121, 1263–1272. 2754. Maves, L. and Schubiger, G. (1998). A molecular basis for transdetermination in Drosophila imaginal discs: interactions between wingless and decapentaplegic signaling. Development 125, 115–124. 2755. Maves, L. and Schubiger, G. (1999). Cell determination and transdetermination in Drosophila imaginal discs. Curr. Topics Dev. Biol. 43, 115–151. 2756. Mayer, B.J. (1999). Endocytosis: EH domains lend a hand. Curr. Biol. 9, R70–R73. 2757. Mayer, B.J. and Eck, M.J. (1995). Minding your p’s and q’s. Curr. Biol. 5, 364–367. 2758. Mayer, B.J., Hirai, H., and Sakai, R. (1995). Evidence that SH2 domains promote processive phosphorylation by protein-tyrosine kinases. Curr. Biol. 5, 296–305. 2759. Mayer, B.J., Ren, R., Clark, K.L., and Baltimore, D. (1993). A putative modular domain present in diverse signaling proteins. Cell 73, 629–630. 2760. Mayer, M.P. and Bukau, B. (1999). Molecular chaperones: the busy life of Hsp90. Curr. Biol. 9, R322–R325. 2761. Mayer-Jaekel, R.E., Ohkura, H., Gomes, R., Sunkel, C.E., Baumgartner, S., Hemmings, B.A., and Glover, D.M. (1993). The 55 kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase. Cell 72, 621– 633. 2762. Maynard Smith, J. (1968). The counting problem. In “Towards a Theoretical Biology. I. Prolegomena,” (C.H. Waddington, ed.). Aldine Pub. Co.: Chicago, pp. 120–124. 2763. Maynard Smith, J. (1970). Natural selection and the concept of a protein space. Nature 225, 563–564. 2764. Maynard Smith, J. (1983). Evolution and development. In “Development and Evolution,” Symp. Brit. Soc. Dev. Biol., Vol. 6 (B.C. Goodwin, N. Holder, and C.C. Wylie, eds.). Cambridge Univ. Pr.: Cambridge, pp. 33–45. 2765. Maynard Smith, J. (1998). “Shaping Life: Genes, Embryos and Evolution.” Yale Univ. Pr., New Haven. 2766. Maynard Smith, J. (1999). The idea of information in biology. Quart. Rev. Biol. 74, 395–400. 2767. Maynard Smith, J., Burian, R., Kauffman, S., Alberch, P., Campbell, J., Goodwin, B., Lande, R., Raup, D., and Wolpert, L. (1985). Developmental constraints and evolution. Q. Rev. Biol. 60, 265–287. 2768. Maynard Smith, J. and Sondhi, K.C. (1960). The genetics of a pattern. Genetics 45, 1039–1050. 2769. Maynard Smith, J. and Sondhi, K.C. (1961). The arrangement of bristles in Drosophila. J. Embryol. Exp. Morph. 9, 661–672. 2770. McAdams, H.H. and Shapiro, L. (1995). Circuit simulation of genetic networks. Science 269, 650–656. 2771. McCabe, J., French, V., and Partridge, L. (1997). Joint regulation of cell size and cell number in the wing blade of Drosophila melanogaster. Genet. Res. 69, 61–68. 2772. McCall, K. and Bender, W. (1996). Probes for chromatin accessibility in the Drosophila bithorax complex respond differently to Polycomb-mediated repression. EMBO J. 15, 569–580. 2773. McCall, K., O’Connor, M.B., and Bender, W. (1994). Enhancer traps in the Drosophila bithorax complex mark parasegmental domains. Genetics 138, 387–399. 2774. McCall, K. and Steller, H. (1997). Facing death in the ﬂy: genetic analysis of apoptosis in Drosophila. Trends Genet. 13, 222–226. 2775. McCartney, B.M., Dierick, H.A., Kirkpatrick, C., Moline,

REFERENCES

2776.

2777.

2778.

2779.

2780.

2781. 2782. 2783.

2784. 2785.

2786. 2787.

2788. 2789.

2790. 2791. 2792.

2793. 2794.

2795.

M.M., Baas, A., Peifer, M., and Bejsovec, A. (1999). Drosophila APC2 is a cytoskeletally-associated protein that regulates Wingless signaling in the embryonic epidermis. J. Cell Biol. 146, 1303–1318. McCartney, B.M., Kulikauskas, R.M., LaJeunesse, D.R., and Fehon, R.G. (2000). The Neuroﬁbromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation. Development 127, 1315–1324. McCarty, J.H. (1998). The Nck SH2/SH3 adaptor protein: a regulator of multiple intracellular signal transduction events. BioEssays 20, 913–921. McCormick, A., Cor´e, N., Kerridge, S., and Scott, M.P. (1995). Homeotic response elements are tightly linked to tissue-speciﬁc elements in a transcriptional enhancer of the teashirt gene. Development 121, 2799–2812. McDowell, N. and Gurdon, J.B. (1999). Activin as a morphogen in Xenopus mesoderm induction. Sems. Cell Dev. Biol. 10, 311–317. McDowell, N., Zorn, A.M., Crease, D.J., and Gurdon, J.B. (1997). Activin has direct long-range signalling activity and can form a concentration gradient by diffusion. Curr. Biol. 7, 671–681. McEwen, D.G. and Peifer, M. (2000). Wnt signaling: Moving in a new direction. Curr. Biol. 10, R562–R564. McGinnis, W. (1994). A century of homeosis, a decade of homeoboxes. Genetics 137, 607–611. McGinnis, W., Garber, R.L., Wirz, J., Kuroiwa, A., and Gehring, W.J. (1984). A homologous protein-coding sequence in Drosophila homeotic genes and its conservation in other metazoans. Cell 37, 403–408. McGinnis, W. and Krumlauf, R. (1992). Homeobox genes and axial patterning. Cell 68, 283–302. McGinnis, W., Levine, M.S., Hafen, E., Kuroiwa, A., and Gehring, W.J. (1984). A conserved DNA sequence in homoeotic genes of the Drosophila Antennapedia and bithorax complexes. Nature 308, 428–433. McGrew, M.J. and Pourqui´e, O. (1998). Somitogenesis: segmenting a vertebrate. Curr. Opin. Gen. Dev. 8, 487–493. McIver, S.B. (1985). Mechanoreception. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology,” Vol. 6 (G.A. Kerkut and L.I. Gilbert, eds.). Pergamon: New York, pp. 71–132. McKinney, M.L., ed. (1988). “Heterochrony in Evolution: A Multidisciplinary Approach.” Plenum Pr., New York. McKinney, M.L. and McNamara, K.J. (1991). “Heterochrony: The Evolution of Ontogeny.” Plenum Pr., New York. McKnight, S.L. (1991). Molecular zippers in gene regulation. Sci. Am. 264 #4, 54–64. McMahon, A.P. (2000). More surprises in the Hedgehog signaling pathway. Cell 100, 185–188. McNeill, H. (2000). Sticking together and sorting things out: adhesion as a force in development. Nature Rev. Gen. 1, 100–108. McNeill, H. and Downward, J. (1999). Apoptosis: Ras to the rescue in the ﬂy eye. Curr. Biol. 9, R176–R179. McNeill, H., Yang, C.-H., Brodsky, M., Ungos, J., and Simon, M.A. (1997). mirror encodes a novel PBX-class homeoprotein that functions in the deﬁnition of the dorsal-ventral border in the Drosophila eye. Genes Dev. 11, 1073–1082. McNiven, M.A., Cao, H., Pitts, K.R., and Yoon, Y. (2000). The dynamin family of mechanoenzymes: pinching in new places. Trends Bioch. Sci. 25, 115–120. [See also 2001 Curr. Biol. 11, R850.]

REFERENCES

2796. Mechler, B.M., McGinnis, W., and Gehring, W. (1985). Molecular cloning of lethal(2)giant larvae, a recessive oncogene of Drosophila melanogaster. EMBO J. 4, 1551– 1557. 2797. Meier, P. and Evan, G. (1998). Dying like ﬂies. Cell 95, 295– 298. 2798. Meinertzhagen, I.A. (1975). The development of neuronal connection patterns in the visual systems of insects. In “Cell Patterning,” Ciba Found. Symp., Vol. 29 (R. Porter and J. Rivers, eds.). Elsevier: Amsterdam, pp. 265–288. [Cf. 2001 Neuron 32, 225–235, 237–248, 381–384.] 2799. Meinertzhagen, I.A. (1993). Sleeping neuroblasts: the proliferation of optic lobe stem cells in Drosophila is regulated by extrinsic factors that include short-range interactions with ingrowing retinal axons and glial cells. Curr. Biol. 3, 904–906. 2800. Meinertzhagen, I.A. (2000). Wiring the ﬂy’s eye. Neuron 28, 310–313. 2801. Meinertzhagen, I.A. and Hanson, T.E. (1993). The development of the optic lobe. In “The Development of Drosophila melanogaster,” Vol. 2 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Lab. Pr.: Plainview, N. Y., pp. 1363– 1491. 2802. Meinhardt, H. (1978). Models for the ontogenetic development of higher organisms. Rev. Physiol. Biochem. Pharmacol. 80, 47–104. 2803. Meinhardt, H. (1978). Space-dependent cell determination under the control of a morphogen gradient. J. Theor. Biol. 74, 307–321. 2804. Meinhardt, H. (1980). Cooperation of compartments for the generation of positional information. Z. Naturforsh. 35c, 1086–1091. 2805. Meinhardt, H. (1982). Generation of structures in a developing organism. In “Developmental Order: Its Origin and Regulation,” (S. Subtelny and P.B. Green, eds.). Alan R. Liss: New York, pp. 439–461. 2806. Meinhardt, H. (1982). “Models of Biological Pattern Formation.” Acad. Pr., New York. 2807. Meinhardt, H. (1982). The role of compartmentalization in the activation of particular control genes and in the generation of proximo-distal positional information in appendages. Amer. Zool. 22, 209–220. 2808. Meinhardt, H. (1983). Cell determination boundaries as organizing regions for secondary embryonic ﬁelds. Dev. Biol. 96, 375–385. 2809. Meinhardt, H. (1984). Models for pattern formation during development of higher organisms. In “Pattern Formation: A Primer in Developmental Biology,” (G.M. Malacinski and S.V. Bryant, eds.). Macmillan: New York, pp. 47–72. 2810. Meinhardt, H. (1984). Models for positional signaling, the threefold subdivision of segments and the pigmentation pattern of molluscs. J. Embryol. Exp. Morph. 83 (Suppl.), 289–311. 2811. Meinhardt, H. (1986). Hierarchical inductions of cell states: a model for segmentation in Drosophila. J. Cell Sci. Suppl. 4, 357–381. 2812. Meinhardt, H. (1986). The threefold subdivision of segments and the initiation of legs and wings in insects. Trends Genet. 2, 36–41. 2813. Meinhardt, H. (1991). Determination borders as organizing regions in the generation of secondary embryonic ﬁelds: the initiation of legs and wings. Sems. Dev. Biol. 2, 129–138. 2814. Meinhardt, H. (1994). Biological pattern formation: new

383

2815.

2816.

2817.

2818.

2819.

2820.

2821.

2822.

2823.

2824.

2825.

2826. 2827.

2828.

2829.

2830.

2831.

observations provide support for theoretical predictions. BioEssays 16, 627–632. Meinhardt, H. and Gierer, A. (1974). Applications of a theory of biological pattern formation based on lateral inhibition. J. Cell Sci. 15, 321–346. Meinhardt, H. and Gierer, A. (1980). Generation and regeneration of sequence of structures during morphogenesis. J. Theor. Biol. 85, 429–450. Meinhardt, H. and Gierer, A. (1981). Generation of spatial sequences of structures during development of higher organisms. In “Lectures on Mathematics in the Life Sciences,” Vol. 14 Am. Math. Soc.: Providence, Rhode Island, pp. 1–20. Meinhardt, H. and Gierer, A. (2000). Pattern formation by local self-activation and lateral inhibition. BioEssays 22, 753–760. Meise, M. and Janning, W. (1993). Cell lineage of larval and imaginal thoracic anlagen cells of Drosophila melanogaster, as revealed by single-cell transplantations. Development 118, 1107–1121. Meise, M. and Janning, W. (1994). Localization of thoracic imaginal-disc precursor cells in the early embryo of Drosophila melanogaster. Mechs. Dev. 48, 109–117. Meisner, H., Daga, A., Buxton, J., Fern´andez, B., Chawla, A., Banerjee, U., and Czech, M.P. (1997). Interactions of Drosophila Cbl with epidermal growth factor receptors and role of Cbl in R7 photoreceptor cell development. Mol. Cell. Biol. 17, 2217–2225. Mel´endez, A., Li, W., and Kalderon, D. (1995). Activity, expression and function of a second Drosophila protein kinase A catalytic subunit gene. Genetics 141, 1507–1520. Melnick, M.B., Perkins, L.A., Lee, M., Ambrosio, L., and Perrimon, N. (1993). Developmental and molecular characterization of mutations in the Drosophila-raf serine/threonine protein kinase. Development 118, 127– 138. Melzer, R.R., Sprenger, J., Nicastro, D., and Smola, U. (1999). Larva-adult relationships in an ancestral dipteran: a re-examination of sensillar pathways across the antenna and leg anlagen of Chaoborus crystallinus (DeGeer, 1776; Chaoboridae). Dev. Genes Evol. 209, 103–112. Mercader, N., Leonardo, E., Azpiazu, N., Serrano, A., Morata, G., Mart´ınez-A, C., and Torres, M. (1999). Conserved regulation of proximodistal limb axis development by Meis1/Hth. Nature 402, 425–429. Merika, M. and Thanos, D. (2001). Enhanceosomes. Curr. Opin. Gen. Dev. 11, 205–208. Merli, C., Bergstrom, D.E., Cygan, J.A., and Blackman, R.K. (1996). Promoter speciﬁcity mediates the independent regulation of neighboring genes. Genes Dev. 10, 1260– 1270. [See also 2001 Genes Dev. 15, 2515–2519.] Merriam, J.R. (1978). Estimating primordial cell numbers in Drosophila imaginal discs and histoblasts. In “Genetic Mosaics and Cell Differentiation,” Results and Problems in Cell Differentiation, Vol. 9, (W.J. Gehring, ed.). SpringerVerlag: Berlin, pp. 71–96. Merrill, V.K.L., Diederich, R.J., Turner, F.R., and Kaufman, T.C. (1989). A genetic and developmental analysis of mutations in labial, a gene necessary for proper head formation in Drosophila melanogaster. Dev. Biol. 135, 376–391. Merrill, V.K.L., Turner, F.R., and Kaufman, T.C. (1987). A genetic and developmental analysis of mutations in the Deformed locus in Drosophila melanogaster. Dev. Biol. 122, 379–395. Merritt, D.J., Hawken, A., and Whitington, P.M. (1993). The

384

2832.

2833.

2834.

2835.

2836. 2837. 2838.

2839.

2840. 2841.

2842. 2843.

2844.

2845.

2846. 2847.

2848.

2849.

2850.

2851.

REFERENCES

role of the cut gene in the speciﬁcation of central projections by sensory axons in Drosophila. Neuron 10, 741–752. M´ethot, N. and Basler, K. (1999). Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Cell 96, 819–831. [See also 2001 Development 128, 4361–4370.] M´ethot, N. and Basler, K. (2000). Suppressor of fused opposes Hedgehog signal transduction by impeding nuclear accumulation of the activator form of Cubitus interruptus. Development 127, 4001–4010. M´ethot, N. and Basler, K. (2001). An absolute requirement for Cubitus interruptus in Hedgehog signaling. Development 128, 733–742. Meyer-Rochow, V.B. (2000). The eye: monophyletic, polyphyletic or perhaps biphyletic? Trends Genet. 16, 244–245 (cf. Gehring’s response). Meyerowitz, E.M. (1997). Plants and the logic of development. Genetics 145, 5–9. Mglinets, V.A. and Kostina, I.V. (1978). Genetic control of bristle length in Drosophila. Genetika 14, 285–293. Micchelli, C.A. and Blair, S.S. (1999). Dorsoventral lineage restriction in wing imaginal discs requires Notch. Nature 401, 473–476. [See also 2001 references: Cell 106, 785–794; Curr. Biol. 11, R1017–R1021.] Micchelli, C.A., Rulifson, E.J., and Blair, S.S. (1997). The function and regulation of cut expression on the wing margin of Drosophila: Notch, Wingless and a dominant negative role for Delta and Serrate. Development 124, 1485– 1495. Michaelson, J. (1987). Cell selection in development. Biol. Rev. 62, 115–139. Michaely, P. and Bennett, V. (1992). The ANK repeat: a ubiquitous motif involved in macromolecular recognition. Trends Cell Biol. 2, 127–129. Michelson, A.M. (1996). A new turn (or two) for Twist. Science 272, 1449–1450. Middleton, J., Neil, S., Wintle, J., Clark-Lewis, I., Moore, H., Lam, C., Auer, M., Hub, E., and Rot, A. (1997). Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell 91, 385–395. Mihaly, J., Hogga, I., Gausz, J., Gyurkovics, H., and Karch, F. (1997). In situ dissection of the Fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycomb-response element. Development 124, 1809– 1820. Miklos, G.L.G. and Rubin, G.M. (1996). The role of the Genome Project in determining gene function: insights from model organisms. Cell 86, 521–529. Mil´an, M. (1998). Cell cycle control in the Drosophila wing. BioEssays 20, 969–971. Mil´an, M., Baonza, A., and Garc´ıa-Bellido, A. (1997). Wing surface interactions in venation patterning in Drosophila. Mechs. Dev. 67, 203–213. Mil´an, M., Campuzano, S., and Garc´ıa-Bellido, A. (1996). Cell cycling and patterned cell proliferation in the wing primordium of Drosophila. Proc. Natl. Acad. Sci. USA 93, 640–645. Mil´an, M., Campuzano, S., and Garc´ıa-Bellido, A. (1997). Developmental parameters of cell death in the wing disc of Drosophila. Proc. Natl. Acad. Sci. USA 94, 5691–5696. Mil´an, M. and Cohen, S.M. (1999). Notch signaling is not sufﬁcient to deﬁne the afﬁnity boundary between dorsal and ventral compartments. Molec. Cell 4, 1073–1078. Mil´an, M. and Cohen, S.M. (1999). Regulation of LIM

2852.

2853.

2854.

2855.

2856.

2857.

2858.

2859.

2860.

2861.

2862.

2863.

2864.

2865.

2866.

2867.

2868.

homeodomain activity in vivo: a tetramer of dLDB and Apterous confers activity and capacity for regulation by dLMO. Molec. Cell 4, 267–273. Mil´an, M. and Cohen, S.M. (2000). Subdividing cell populations in the developing limbs of Drosophila: Do wing veins and leg segments deﬁne units of growth control? Dev. Biol. 217, 1–9. Mil´an, M. and Cohen, S.M. (2000). Temporal regulation of Apterous activity during development of the Drosophila wing. Development 127, 3069–3078. Mil´an, M., Diaz-Benjumea, F.J., and Cohen, S.M. (1998). Beadex encodes an LMO protein that regulates Apterous LIM-homeodomain activity in Drosophila wing development: a model for LMO oncogene function. Genes Dev. 12, 2912–2920. ¨ Milburn, M.V., Tong, L., deVos, A.M., Brunger, A., Yamaizumi, Z., Nishimura, S., and Kim, S.-H. (1990). Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science 247, 939–945. Mil´etich, I. and Limbourg-Bouchon, B. (2000). Drosophila null slimb clones transiently deregulate Hedgehogindependent transcription of wingless in all limb discs, and induce decapentaplegic transcription linked to imaginal disc regeneration. Mechs. Dev. 93, 15–26. Miller, A. (1950). The internal anatomy and histology of the imago of Drosophila melanogaster. In “Biology of Drosophila,” (M. Demerec, ed.). Hafner: New York, pp. 420–534. Miller, C. and Blair, S. (2001). Genetic interactions between shifted and members of the Hedgehog signaling pathway. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a188. Miller, D.T. and Cagan, R.L. (1998). Local induction of patterning and programmed cell death in the developing Drosophila retina. Development 125, 2327–2335. Miller, J.R. and Moon, R.T. (1996). Signal transduction through β-catenin and speciﬁcation of cell fate during embryogenesis. Genes Dev. 10, 2527–2539. Millward, T.A., Zolnierowicz, S., and Hemmings, B.A. (1999). Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem. Sci. 24, 186–191. Milner, M.J., Bleasby, A.J., and Kelly, S.L. (1984). The role of the peripodial membrane of leg and wing imaginal discs of Drosophila melanogaster during evagination and differentiation in vitro. Roux’s Arch. Dev. Biol. 193, 180– 186. Milner, M.J., Bleasby, A.J., and Pyott, A. (1983). The role of the peripodial membrane in the morphogenesis of the eye-antennal disc of Drosophila melanogaster. Roux’s Arch. Dev. Biol. 192, 164–170. Milner, M.J., Bleasby, A.J., and Pyott, A. (1984). Cell interactions during the fusion in vitro of Drosophila eye-antennal imaginal discs. Roux’s Arch. Dev. Biol. 193, 406–413. Milner, M.J. and Haynie, J.L. (1979). Fusion of Drosophila eye-antennal imaginal discs during differentiation in vitro. W. Roux’s Arch. 185, 363–370. Milton, C.C., McKenzie, J.A., Woods, R.E., and Batterham, P. (2001). Effect of chaperone genes on asymmetry. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a269. Minami, M., Kinoshita, N., Kamoshida, Y., Tanimoto, H., and Tabata, T. (1999). brinker is a target of Dpp in Drosophila that negatively regulates Dpp-dependent genes. Nature 398, 242–246. Minelli, A. (2000). Limbs and tail as evolutionarily diverging duplicates of the main body axis. Evol. Dev. 2, 157–165.

REFERENCES

2869. Minelli, A. and Bortoletto, S. (1988). Myriapod metamerism and arthropod segmentation. Biol. J. Linnean Soc. 33, 323–343. [Cf. 2001 Dev. Genes Evol. 211, 509–521.] 2870. Minsky, M., ed. (1968). “Semantic Information Processing.” M.I.T. Pr., Cambridge, Mass. [See also 2001 Am. Sci. 89, 204–208.] 2871. Minsky, M. (1985). “The Society of Mind.” Simon & Schuster, New York. 2872. Mirkovic, I., Smith, J.L., Gorski, S.M., and Verheyen, E.M. (2001). Nemo functions at multiple stages during Drosophila epidermal development. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a174. [See also 2001 Mechs. Dev. 101, 119–132.] 2873. Miskiewicz, P., Morrissey, D., Lan, Y., Raj, L., Kessler, S., Fujioka, M., Goto, T., and Weir, M. (1996). Both the paired domain and homeodomain are required for in vivo function of Drosophila Paired. Development 122, 2709– 2718. ¨ 2874. Missler, M. and Sudhof, T.C. (1998). Neurexins: three genes and 1001 products. Trends Genet. 14, 20–26. 2875. Misteli, T. (2001). Protein dynamics: implications for nuclear architecture and gene expression. Science 291, 843– 847. 2876. Mitchell, H.K., Edens, J., and Petersen, N.S. (1990). Stages of cell hair construction in Drosophila. Dev. Genet. 11, 133– 140. 2877. Mitchell, H.K. and Petersen, N.S. (1989). Epithelial differentiation in Drosophila pupae. Dev. Genet. 10, 42–52. 2878. Mitchell, P.J. and Tjian, R. (1989). Transcriptional regulation in mammalian cells by sequence-speciﬁc DNA binding proteins. Science 245, 371–378. 2879. Mitchelson, A., Simonelig, M., Williams, C., and O’Hare, K. (1993). Homology with Saccharomyces cerevisiae RNA14 suggests that phenotypic suppression in Drosophila melanogaster by suppressor of forked occurs at the level of RNA stability. Genes Dev. 7, 241–249. 2880. Mittal, R., Ahmadian, M.R., Goody, R.S., and Wittinghofer, A. (1996). Formation of a transition-state analog of the Ras GTPase reaction by RAS.GDP, tetraﬂuoroaluminate, and GTPase-activating proteins. Science 273, 115– 117. 2881. Mittenthal, J.E. (1981). The rule of normal neighbors: A hypothesis for morphogenetic pattern regulation. Dev. Biol. 88, 15–26. 2882. Miyamoto, H., Nihonmatsu, I., Kondo, S., Ueda, R., Togashi, S., Hirata, K., Ikegami, Y., and Yamamoto, D. (1995). canoe encodes a novel protein containing a GLGF/DHR motif and functions with Notch and scabrous in common developmental pathways in Drosophila. Genes Dev. 9, 612– 625. 2883. Mlodzik, M. (1999). Planar polarity in the Drosophila eye: a multifaceted view of signaling speciﬁcity and cross-talk. EMBO J. 18, 6873–6879. 2884. Mlodzik, M. (2000). Spiny legs and prickled bodies: new insights and complexities in planar polarity establishment. BioEssays 22, 311–315. 2885. Mlodzik, M., Baker, N.E., and Rubin, G.M. (1990). Isolation and expression of scabrous, a gene regulating neurogenesis in Drosophila. Genes Dev. 4, 1848–1861. 2886. Mlodzik, M., Gibson, G., and Gehring, W.J. (1990). Effects of ectopic expression of caudal during Drosophila development. Development 109, 271–277. 2887. Mlodzik, M., Hiromi, Y., Goodman, C.S., and Rubin, G.M. (1992). The presumptive R7 cell of the developing Drosophila eye receives positional information indepen-

385

dent of sevenless, boss and sina. Mechs. Dev. 37, 37–42. 2888. Mlodzik, M., Hiromi, Y., Weber, U., Goodman, C.S., and Rubin, G.M. (1990). The Drosophila seven-up gene, a member of the steroid receptor gene superfamily, controls photoreceptor cell fate. Cell 60, 211–224. 2889. Moazed, D. and O’Farrell, P.H. (1992). Maintenance of the engrailed expression pattern by Polycomb group genes in Drosophila. Development 116, 805–810. 2890. Modolell, J. (1997). Patterning of the adult peripheral nervous system of Drosophila. Persp. Dev. Neurobiol. 4, 285– 296. 2891. Modolell, J. and Campuzano, S. (1998). The achaete-scute complex as an integrating device. Int. J. Dev. Biol. 42, 275– 282. 2892. Moghal, N. and Sternberg, P.W. (1999). Multiple positive and negative regulators of signaling by the EGF receptor. Curr. Opin. Cell Biol. 11, 190–196. 2893. Mohler, J. (1988). Requirements for hedgehog, a segmental polarity gene, in patterning larval and adult cuticle of Drosophila. Genetics 120, 1061–1072. 2894. Mohler, J., Seecoomar, M., Agarwal, S., Bier, E., and Hsai, J. (2000). Activation of knot (kn) speciﬁes the 3-4 intervein region in the Drosophila wing. Development 127, 55– 63. 2895. Mohler, J. and Vani, K. (1992). Molecular organization and embryonic expression of the hedgehog gene involved in cell-cell communication in segmental patterning of Drosophila. Development 115, 957–971. 2896. Mohler, J.D. (1965). The inﬂuence of some crossveinlesslike genes on the crossveinless phenocopy sensitivity in Drosophila melanogaster. Genetics 51, 329–340. 2897. Mohler, J.D. (1965). Preliminary genetic analysis of crossveinless-like strains of Drosophila melanogaster. Genetics 51, 641–651. 2898. Mohler, J.D. and Swedberg, G.S. (1964). Wing vein development in crossveinless-like strains of Drosophila melanogaster. Genetics 50, 1403–1419. 2899. Moline, M.M., Dierick, H.A., Southern, C., and Bejsovec, A. (2000). Non-equivalent roles of Drosophila Frizzled and Dfrizzled2 in embryonic Wingless signal transduction. Curr. Biol. 10, 1127–1130. 2900. Moline, M.M., Southern, C., and Bejsovec, A. (1999). Directionality of Wingless protein transport inﬂuences epidermal patterning in the Drosophila embryo. Development 126, 4375–4384. 2901. Møller, A.P. and Swaddle, J.P. (1997). “Asymmetry, Developmental Stability, and Evolution.” Oxford Univ. Pr., Oxford. 2902. Mollereau, B., Dominguez, M., Webel, R., Colley, N.J., Keung, B., De Celis, J.F., and Desplan, C. (2001). Twostep process for photoreceptor formation in Drosophila. Nature 412, 911–913. 2903. Mollereau, B., Wernet, M.F., Beauﬁls, P., Killian, D., ¨ Pichaud, F., Kuhnlein, R., and Desplan, C. (2000). A green ﬂuorescent protein enhancer trap screen in Drosophila photoreceptor cells. Mechs. Dev. 93, 151–160. 2904. Moloney, D.J., Panin, V.M., Johnston, S.H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K.D., Haltiwanger, R.S., and Vogt, T.F. (2000). Fringe is a glycosyltransferase that modiﬁes Notch. Nature 406, 369– 375. 2905. Monge, I. and Mitchell, P.J. (1998). DAP-2, the Drosophila homolog of transcription factor AP-2. Mechs. Dev. 76, 191– 195. 2906. Monnier, V., Dussillol, F., Alves, G., Lamour-Isnard, C., and Plessis, A. (1998). Suppressor of fused links Fused and

386

2907.

2908. 2909. 2910. 2911. 2912.

2913. 2914.

2915.

2916.

2917. 2918. 2919.

2920. 2921.

2922. 2923. 2924.

2925. 2926.

2927.

2928.

2929.

REFERENCES

Cubitus interruptus on the Hedgehog signalling pathway. Curr. Biol. 8, 583–586. Montagne, J., Groppe, J., Guillemin, K., Krasnow, M.A., Gehring, W.J., and Affolter, M. (1996). The Drosophila Serum Response Factor gene is required for the formation of intervein tissue of the wing and is allelic to blistered. Development 122, 2589–2597. Montell, C. (1989). Molecular genetics of Drosophila vision. BioEssays 11, 43–48. Montell, C. (1999). Visual transduction in Drosophila. Annu. Rev. Cell Dev. Biol. 15, 231–268. Montminy, M. (1993). Trying on a new pair of SH2s. Science 261, 1694–1695. Montminy, M. (1997). Something new to hang your HAT on. Nature 387, 654–655. Moodie, S.A., Willumsen, B.M., Weber, M.J., and Wolfman, A. (1993). Complexes of Ras.GTP with Raf-1 and mitogenactivated protein kinase kinase. Science 260, 1658–1661. Moodie, S.A. and Wolfman, A. (1994). The 3 Rs of life: Ras, Raf and growth regulation. Trends Genet. 10, 44–48. Moon, R.T. and Miller, J.R. (1997). The APC tumor suppressor protein in development and cancer. Trends Genet. 13, 256–258. Moore, B.W. and McGregor, D. (1965). Chromatographic and electrophoretic fractionation of soluble proteins of brain and liver. J. Biol. Chem. 240 #4, 1647–1653. Moore, B.W. and Perez, V.J. (1968). Speciﬁc acidic proteins of the nervous system. In “Physiological and Biochemical Aspects of Nervous Integration,” (F.D. Carlson, ed.). Prentice-Hall: Englewood Cliffs, N. J., pp. 343–359. Moore, J.A. (1986). Science as a way of knowing–genetics. Amer. Zool. 26, 583–747. Moore, J.A. (1987). Science as a way of knowing– developmental biology. Amer. Zool. 27, 415–573. Moore, J.A. (1993). “Science as a Way of Knowing. The Foundations of Modern Biology.” Harvard Univ. Pr., Cambridge, Mass. Moore, P.B. (1999). Structural motifs in RNA. Annu. Rev. Biochem. 67, 287–300. Morata, G. (1975). Analysis of gene expression during development in the homeotic mutant Contrabithorax of Drosophila melanogaster. J. Embryol. Exp. Morph. 34, 19–31. Morata, G. (1982). The mode of action of the bithorax genes of Drosophila melanogaster. Amer. Zool. 22, 57–64. Morata, G. (1993). Homeotic genes of Drosophila. Curr. Opin. Gen. Dev. 3, 606–614. Morata, G. and Garcia-Bellido, A. (1976). Developmental analysis of some mutants of the bithorax system of Drosophila. W. Roux’s Arch. 179, 125–143. Morata, G. and Kerridge, S. (1981). Sequential functions of the bithorax complex of Drosophila. Nature 290, 778–781. Morata, G. and Kerridge, S. (1982). The role of position in determining homoeotic gene function in Drosophila. Nature 300, 191–192. Morata, G., Kornberg, T., and Lawrence, P.A. (1983). The phenotype of engrailed mutations in the antenna of Drosophila. Dev. Biol. 99, 27–33. Morata, G. and Lawrence, P.A. (1975). Control of compartment development by the engrailed gene in Drosophila. Nature 255, 614–617. Morata, G. and Lawrence, P.A. (1977). The development of wingless, a homeotic mutation of Drosophila. Dev. Biol. 56, 227–240.

2930. Morata, G. and Lawrence, P.A. (1977). Homoeotic genes, compartments and cell determination in Drosophila. Nature 265, 211–216. 2931. Morata, G. and Lawrence, P.A. (1978). Anterior and posterior compartments in the head of Drosophila. Nature 274, 473–474. 2932. Morata, G. and Lawrence, P.A. (1978). Cell lineage and homeotic mutants in the development of imaginal discs of Drosophila. In “The Clonal Basis of Development,” Symp. Soc. Dev. Biol., Vol. 36 (S. Subtelny and I.M. Sussex, eds.). Acad. Pr.: New York, pp. 45–60. 2933. Morata, G. and Lawrence, P.A. (1979). Development of the eye-antenna imaginal disc of Drosophila. Dev. Biol. 70, 355–371. 2934. Morata, G., Mac´ıas, A., Urqu´ıa, N., and Gonz´alez-Reyes, A. (1990). Homoeotic genes. Sems. Cell Biol. 1, 219–227. 2935. Morata, G. and Ripoll, P. (1975). Minutes: mutants of Drosophila autonomously affecting cell division rate. Dev. Biol. 42, 211–221. 2936. Morata, G. and S´anchez-Herrero, E. (1998). Pulling the ﬂy’s leg. Nature 392, 657–658. 2937. Morata, G. and S´anchez-Herrero, E. (1999). Patterning mechanisms in the body trunk and the appendages of Drosophila. Development 126, 2823–2828. 2938. Morata, G. and Struhl, G. (1990). Fly ﬁshing downstream. Nature 348, 587–588. 2939. Morcillo, P., Rosen, C., Baylies, M.K., and Dorsett, D. (1997). Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila. Genes Dev. 11, 2729–2740. 2940. Morcillo, P., Rosen, C., and Dorsett, D. (1996). Genes regulating the remote wing margin enhancer in the Drosophila cut locus. Genetics 144, 1143–1154. 2941. Morel, V. and Schweisguth, F. (2000). Repression by Suppressor of Hairless and activation by Notch are required to deﬁne a single row of single-minded expressing cells in the Drosophila embryo. Genes Dev. 14, 377–388. 2942. Moreno, E. and Morata, G. (1999). Caudal is the Hox gene that speciﬁes the most posterior Drosophila segment. Nature 400, 873–877. 2943. Morgan, D.O. (1997). Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu. Rev. Cell Dev. Biol. 13, 261–291. 2944. Morgan, R., In der Rieden, P., Hooiveld, M.H.W., and Durston, A.J. (2000). Identifying HOX paralog groups by the PBX-binding region. Trends Genet. 16, 66–67. 2945. Morgan, T.H. (1901). “Regeneration.” MacMillan, New York. 2946. Morgan, T.H. (1910). Chance or purpose in the origin and evolution of adaptation. Science 31, 201–210. 2947. Morgan, T.H. (1910). Chromosomes and heredity. Am. Nat. 44, 449–496. 2948. Morgan, T.H. (1910). Sex limited inheritance in Drosophila. Science 32, 120–122. 2949. Morgan, T.H. (1934). “Embryology and Genetics.” Greenwood Pr., Westport, Conn. 2950. Morgan, T.H. and Bridges, C.B. (1919). The origin of gynandromorphs. In “Contributions to the Genetics of Drosophila melanogaster,” Vol. Pub. No. 278 Carnegie Inst. Wash.: Wash. D. C., pp. 1–122. 2951. Morgan, T.H., Bridges, C.B., and Sturtevant, A.H. (1925). The Genetics of Drosophila. Bibliogr. Genet. 2, 1–262. 2952. Morimoto, A.M., Jordan, K.C., Tietze, K., Britton, J.S., O’Neill, E.M., and Ruohola-Baker, H. (1996). Pointed, an

REFERENCES

2953.

2954.

2955.

2956.

2957. 2958.

2959.

2960.

2961. 2962. 2963.

2964.

2965. 2966.

2967.

2968.

2969.

2970.

2971.

ETS domain transcription factor, negatively regulates the EGF receptor pathway in Drosophila oogenesis. Development 122, 3745–3754. Morimoto, R.I. (1998). Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev. 12, 3788–3796. Morimura, S., Maves, L., and Hoffmann, F.M. (1996). decapentaplegic overexpression affects Drosophila wing and leg imaginal disc development and wingless expression. Dev. Biol. 177, 136–151. Morita, H. and Shiraishi, A. (1985). Chemoreception physiology. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology,” Vol. 6 (G.A. Kerkut and L.I. Gilbert, eds.). Pergamon: New York, pp. 133–170. Moritz, K.B. and Sauer, H.W. (1996). Boveri’s contributions to developmental biology–a challenge for today. Int. J. Dev. Biol. 40, 27–47. Morrison, D.K. and Cutler, R.E., Jr. (1997). The complexity of Raf-1 regulation. Curr. Opin. Cell Biol. 9, 174–179. Morrow, E.M., Furukawa, T., Lee, J.E., and Cepko, C.L. (1999). NeuroD regulates multiple functions in the developing neural retina in rodent. Development 126, 23–36. Moscoso del Prado, J. and Garcia-Bellido, A. (1984). Cell interactions in the generation of chaetae pattern in Drosophila. Roux’s Arch. Dev. Biol. 193, 246–251. Moscoso del Prado, J. and Garcia-Bellido, A. (1984). Genetic regulation of the Achaete-scute complex of Drosophila melanogaster. Roux’s Arch. Dev. Biol. 193, 242– 245. Moses, K. (1991). The role of transcription factors in the developing Drosophila eye. Trends Genet. 7, 250–255. Moses, K., ed. (2002). “Drosophila Eye Development.” Results and Problems in Cell Differentiation, Springer, Berlin. Moses, K., Ellis, M.C., and Rubin, G.M. (1989). The glass gene encodes a zinc-ﬁnger protein required by Drosophila photoreceptor cells. Nature 340, 531–536. Moses, K. and Rubin, G.M. (1991). glass encodes a sitespeciﬁc DNA-binding protein that is regulated in response to positional signals in the developing Drosophila eye. Genes Dev. 5, 583–593. Moss, E.G. (2000). Non-coding RNAs: lightning strikes twice. Curr. Biol. 10, R436–R439. Mottus, R., Sobel, R.E., and Grigliatti, T.A. (2000). Mutational analysis of a histone deacetylase in Drosophila melanogaster: missense mutations suppress gene silencing associated with position effect variegation. Genetics 154, 657–668. Motzny, C.K. and Holmgren, R. (1995). The Drosophila cubitus interruptus protein and its role in the wingless and hedgehog signal transduction pathways. Mechs. Dev. 52, 137–150. Moyer, S.E. and Stepak, S.P. (1971). Inheritance of trident and its role in detecting ebony heterozygotes in D. melanogaster. Dros. Info. Serv. 46, 133. Moyle, W.R., Campbell, R.K., Myers, R.V., Bernard, M.P., Han, Y., and Wang, X. (1994). Co-evolution of ligandreceptor pairs. Nature 368, 251–255. Mozer, B.A. and Benzer, S. (1994). Ingrowth of photoreceptor axons induces transcription of a retrotransposon in the developing Drosophila brain. Development 120, 1049– 1058. Mozer, B.A. and Easwarachandran, K. (1999). Pattern formation in the absence of cell proliferation: tissue-speciﬁc

387

2972.

2973.

2974.

2975.

2976.

2977.

2978. 2979.

2980.

2981.

2982.

2983. 2984.

2985.

2986.

2987.

2988.

2989.

regulation of cell cycle progression by string (stg) during Drosophila eye development. Dev. Biol. 213, 54–69. Muda, M., Boschert, U., Dickinson, R., Martinou, J.-C., Martinou, I., M., C., Schlegel, W., and Arkinstall, S. (1996). MKP-3, a novel cytosolic protein-tyrosine phosphatase that exempliﬁes a new class of mitogen-activated protein kinase phosphatase. J. Biol. Chem. 271, 4319–4326. Mueller, B.K. (1999). Growth cone guidance: ﬁrst steps towards a deeper understanding. Annu. Rev. Neurosci. 22, 351–388. Mugat, B., Brodu, V., Kejzlarova-Lepesant, J., Antoniewski, C., Bayer, C.A., Fristrom, J.W., and Lepesant, J.-A. (2000). Dynamic expression of Broad-Complex isoforms mediates temporal control of an ecdysteroid target gene at the onset of Drosophila metamorphosis. Dev. Biol. 227, 104–117. Mukherjee, A., Lakhotia, S.C., and Roy, J.K. (1995). l(2)gl gene regulates late expression of segment polarity genes in Drosophila. Mechs. Dev. 51, 227–234. Mukherjee, A. and Roy, J.K. (1995). Mutations in tumour suppressor genes, l(2)gl and l(2)gd, alter the expression of wingless in Drosophila. J. Biosci. 20, 333–339. Mukherjee, A., Shan, X., Mutsuddi, M., Ma, Y., and Nambu, J.R. (2000). The Drosophila Sox gene, ﬁsh-hook, is required for postembryonic development. Development 217, 91–106. Mukherjee, A.S. (1965). The effects of sexcombless on the forelegs of Drosophila melanogaster. Genetics 51, 285–304. Mukherjee, A.S. and Hildreth, P.E. (1971). Interaction of the mutants sx, tra and dsx in Drosophila melanogaster. Chaetotaxy of the basitarsus of the forelegs in males and females. Genetica 42, 338–352. ¨ Muller, B. and Basler, K. (2000). The repressor and activator forms of Cubitus interruptus control Hedgehog target genes through common generic Gli-binding sites. Development 127, 2999–3007. ¨ Muller, C. and Leutz, A. (2001). Chromatin remodeling in development and differentiation. Curr. Opin. Gen. Dev. 11, 167–174. ¨ Muller, G.H. (1976). Drosophila: a contribution to its morphology and development by W. F. von Gleichen in 1764. J. Nat. Hist. 10, 581–597. ¨ Muller, H.-A.J. (2000). Genetic control of epithelial cell polarity: lessons from Drosophila. Dev. Dynamics 218, 52–67. ¨ Muller, H.-A.J., Samanta, R., and Wieschaus, E. (1999). Wingless signaling in the Drosophila embryo: zygotic requirements and the role of the frizzled genes. Development 126, 577–586. ¨ Muller, H.-A.J. and Wieschaus, E. (1996). armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila. J. Cell Biol. 134, 149–163. Muller, H.J. (1932). Further studies on the nature and causes of gene mutations. Proc. 6th Internat. Congr. Genet. 1, 213–255. ¨ Muller, J. and Bienz, M. (1991). Long range repression conferring boundaries of Ultrabithorax expression in the Drosophila embryo. EMBO J. 10, 3147–3155. ¨ Muller, M., v. Weizs¨acker, E., and Campos-Ortega, J.A. (1996). Expression domains of a zebraﬁsh homologue of the Drosophila pair-rule gene hairy correspond to primordia of alternating somites. Development 122, 2071–2078. ¨ Muller, W.A. (1996). From the Aristotelian soul to genetic

388

2990. 2991.

2992.

2993.

2994. 2995.

2996.

2997.

2998.

2999.

3000.

3001.

3002.

3003.

3004.

3005.

REFERENCES

and epigenetic information: the evolution of the modern concepts in developmental biology at the turn of the century. Int. J. Dev. Biol. 40, 21–26. Mullins, M.C. (1998). Holy Tolloido: Tolloid cleaves SOG/ Chordin to free DPP/BMPs. Trends Genet. 14, 127–129. Mullins, M.C. and Rubin, G.M. (1991). Isolation of temperature-sensitive mutations of the tyrosine kinase receptor sevenless (sev) in Drosophila and their use in determining its time of action. Proc. Natl. Acad. Sci. USA 88, 9387–9391. Mullor, J.L., Calleja, M., Capdevila, J., and Guerrero, I. (1997). Hedgehog activity, independent of Decapentaplegic, participates in wing disc patterning. Development 124, 1227–1237. Mullor, J.L. and Guerrero, I. (2000). A gain-of-function mutant of patched dissects different responses to the Hedgehog gradient. Dev. Biol. 228, 211–224. Mumm, J.S. and Kopan, R. (2000). Notch signaling: from the outside in. Dev. Biol. 228, 151–165. Mumm, J.S., Schroeter, E.H., Saxena, M.T., Griesemer, A., Tian, X., Pan, D.J., Ray, W.J., and Kopan, R. (2000). A ligandinduced extracellular cleavage regulates γ-secretase-like proteolytic activation of Notch1. Molec. Cell 5, 197–206. Munemitsu, S., Albert, I., Rubinfeld, B., and Polakis, P. (1996). Deletion of an amino-terminal sequence stabilizes β-catenin in vivo and promotes hyperphosphorylation of the adenomatous polyposis coli tumor suppressor protein. Mol. Cell. Biol. 16, 4088–4094. Munemitsu, S., Albert, I., Souza, B., Rubinfeld, B., and Polakis, P. (1995). Regulation of intracellular β-catenin levels by the adenomatous polyposis coli (APC) tumorsuppressor protein. Proc. Natl. Acad. Sci. USA 92, 3046– 3050. Muneoka, K. and Bryant, S. (1984). Regeneration and development of vertebrate appendages. Symp. Zool. Soc. Lond. 52, 177–196. Muneoka, K. and Bryant, S.V. (1982). Evidence that patterning mechanisms in developing and regenerating limbs are the same. Nature 298, 369–371. Munro, S. and Freeman, M. (2000). The Notch signalling regulator Fringe acts in the Golgi apparatus and requires the glycosyltransferase signature motif DxD. Curr. Biol. 10, 813–820. Muravyova, E., Golovnin, A., Gracheva, E., Parshikov, A., Belenkaya, T., Pirrotta, V., and Georgiev, P. (2001). Loss of insulator activity by paired Su(Hw) chromatin insulators. Science 291, 495–498. Muravyova, E., Golovnin, A., Gracheva, E., Pirrota, V., and Georgiev, P. (2001). Paired su(Hw) insulators lose enhancer blocking activity and may instead facilitate enhancerpromoter interactions. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a92. Murayama, M., Tanaka, S., Palacino, J., Murayama, O., Honda, T., Sun, X., Yasutake, K., Nihonmatsu, N., Wolozin, B., and Takashima, A. (1998). Direct association of presenilin-1 with β-catenin. FEBS Letters 433, 73–77. Murphey, R.K., Possidente, D., Pollack, G., and Merritt, D.J. (1989). Modality-speciﬁc axonal projections in the CNS of the ﬂies Phormia and Drosophila. J. Comp. Neurol. 290, 185–200. Murphey, R.K., Possidente, D.R., Vandervorst, P., and Ghysen, A. (1989). Compartments and the topography of leg afferent projections in Drosophila. J. Neurosci. 9, 3209– 3217.

3006. Murphy, C. (1967). Determination of the dorsal mesothoracic disc in Drosophila. Dev. Biol. 15, 368–394. 3007. Murphy, C. and Tokunaga, C. (1970). Cell lineage in the dorsal mesothoracic disc of Drosophila. J. Exp. Zool. 175, 197–219. 3008. Murphy, F.V., IV, Sweet, R.M., and Churchill, M.E.A. (1999). The structure of a chromosomal high mobility group protein-DNA complex reveals sequence-neutral mechanisms important for non-sequence-speciﬁc DNA recognition. EMBO J. 18, 6610–6618. 3009. Murray, A. (1995). Cyclin ubiquitination: the destructive end of mitosis. Cell 81, 149–152. 3010. Murray, A.W. and Kirschner, M.W. (1989). Dominoes and clocks: the union of two views of the cell cycle. Science 246, 614–621. 3011. Murray, J.D. (1981). On pattern formation mechanisms for lepidopteran wing patterns and mammalian coat markings. Phil. Trans. Roy. Soc. Lond. B 295, 473–496. 3012. Murray, J.D. (1981). A pre-pattern formation mechanism for animal coat markings. J. Theor. Biol. 88, 161–199. 3013. Murray, J.D. (1989). “Mathematical Biology.” SpringerVerlag, Berlin. 3014. Murray, J.D. (1990). Turing’s theory of morphogenesis–its inﬂuence on modelling biological pattern and form. Bull. Math. Biol. 52, 119–152. 3015. Murray, M.A., Fessler, L.I., and Palka, J. (1995). Changing distributions of extracellular matrix components during early wing morphogenesis in Drosophila. Dev. Biol. 168, 150–165. 3016. Murre, C., McCaw, P.S., and Baltimore, D. (1989). A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56, 777–783. 3017. Murre, C., McCaw, P.S., Vaessin, H., Caudy, M., Jan, L.Y., Jan, Y.N., Cabrera, C.V., Buskin, J.N., Hauschka, S.D., Lassar, A.B., Weintraub, H., and Baltimore, D. (1989). Interactions between heterologous helix-loop-helix proteins generate complexes that bind speciﬁcally to a common DNA sequence. Cell 58, 537–544. 3018. Murzin, A.G. (1992). Structural principles for the propeller assembly of β-sheets: the preference for seven-fold symmetry. Proteins 14, 191–201. 3019. Musacchio, A., Wilmanns, M., and Saraste, M. (1994). Structure and function of the SH3 domain. Prog. Biophys. Molec. Biol. 61, 283–297. 3020. Musacchio, M. and Perrimon, N. (1996). The Drosophila kekkon genes: novel members of both the leucine-rich repeat and immunoglobulin superfamilies expressed in the CNS. Dev. Biol. 178, 63–76. 3021. Mushegian, A.R. and Koonin, E.V. (1996). Sequence analysis of eukaryotic developmental proteins: ancient and novel domains. Genetics 144, 817–828. 3022. Muskavitch, M.A.T. (1994). Delta-Notch signaling and Drosophila cell fate choice. Dev. Biol. 166, 415–430. 3023. Muslin, A.J., Tanner, J.W., Allen, P.M., and Shaw, A.S. (1996). Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84, 889–897. 3024. Myers, M.P., Wager-Smith, K., Wesley, C.S., Young, M.W., and Sehgal, A. (1995). Positional cloning and sequence analysis of the Drosophila clock gene, timeless. Science 270, 805–808. 3025. Nagaraj, R., Pickup, A.T., Howes, R., Moses, K., Freeman, M., and Banerjee, U. (1999). Role of the EGF receptor pathway in growth and patterning of the Drosophila wing

REFERENCES

3026.

3027.

3028.

3029.

3030. 3031.

3032.

3033.

3034.

3035.

3036.

3037.

3038.

3039.

3040. 3041.

3042.

through the regulation of vestigial. Development 126, 975– 985. Nagata, T., Gupta, V., Sorce, D., Kim, W.-Y., Sali, A., Chait, B.T., Shigesada, K., Ito, Y., and Werner, M.H. (1999). Immunoglobulin motif DNA recognition and heterodimerization of the PEBP2/CBF Runt domain. Nature Struct. Biol. 6, 615–619. Nagel, A.C., Maier, D., and Preiss, A. (2000). Su(H)independent activity of Hairless during mechano-sensory organ formation in Drosophila. Mechs. Dev. 94, 3–12. Nagel, A.C. and Preiss, A. (1999). Notchspl is deﬁcient for inductive processes in the eye, and E(spl)D enhances split by interfering with proneural activity. Dev. Biol. 208, 406–415. Nagel, A.C., Yu, Y., and Preiss, A. (1999). Enhancer of split [E(spl)D ] is a Gro-independent, hypermorphic mutation in Drosophila. Dev. Genet. 25, 168–179. Nagl, W. (1978). “Endopolyploidy and Polyteny in Differentiation and Evolution.” North-Holland, New York. Nagorcka, B.N. (1988). A pattern formation mechanism to control spatial organization in the embryo of Drosophila melanogaster. J. Theor. Biol. 132, 277–306. Nagy, L. (1998). Changing patterns of gene regulation in the evolution of arthropod morphology. Amer. Zool. 38, 818–828. Nakagoshi, H., Hoshi, M., Nabeshima, Y.-i., and Matsuzaki, F. (1998). A novel homeobox gene mediates the Dpp signal to establish functional speciﬁcity within target cells. Genes Dev. 12, 2724–2734. Nakamura, M., Nishida, Y., and Ueno, N. (2001). Genetic screening to isolate novel Dpp signal downstream components. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a163. Nakamura, M., Okano, H., Blendy, J.A., and Montell, C. (1994). Musashi, a neural RNA-binding protein required for Drosophila adult external sensory organ development. Neuron 13, 67–81. [See also 2001 Nature 411, 94–98.] Nakano, Y., Guerrero, I., Hidalgo, A., Taylor, A., Whittle, J.R.S., and Ingham, P.W. (1989). A protein with several possible membrane-spanning domains encoded by the Drosophila segment polarity gene patched. Nature 341, 508–513. Nakao, K. and Campos-Ortega, J.A. (1996). Persistent expression of genes of the Enhancer of Split Complex suppresses neural development in Drosophila. Neuron 16, 275–286. Nakato, H., Futch, T.A., and Selleck, S.B. (1995). The division abnormally delayed (dally) gene: a putative integral membrane proteoglycan required for cell division patterning during postembryonic development of the nervous system in Drosophila. Development 121, 3687–3702. Nakielny, S. and Dreyfuss, G. (1997). Import and export of the nuclear protein import receptor transportin by a mechanism independent of GTP hydrolysis. Curr. Biol. 8, 89–95. Nakielny, S. and Dreyfuss, G. (1999). Transport of proteins and RNAs in and out of the nucleus. Cell 99, 677–690. Namba, R. and Minden, J.S. (1999). Fate mapping of Drosophila embryonic mitotic domain 20 reveals that the larval visual system is derived from a subdomain of a few cells. Dev. Biol. 212, 465–476. Namba, R., Pazdera, T.M., Cerrone, R.L., and Minden, J.S. (1997). Drosophila embryonic pattern repair: how embryos respond to bicoid dosage alteration. Development 124, 1393–1403.

389

3043. Nambu, J.R., Franks, R.G., Hu, S., and Crews, S.T. (1990). The single-minded gene of Drosophila is required for the expression of genes important for the development of CNS midline cells. Cell 63, 63–75. 3044. Nambu, J.R., Lewis, J.O., Wharton, K.A., Jr., and Crews, S.T. (1991). The Drosophila single-minded gene encodes a helix-loop-helix protein that acts as a master regulator of CNS midline development. Cell 67, 1157–1167. 3045. Nambu, P.A. and Nambu, J.R. (1996). The Drosophila ﬁshhook gene encodes a HMG domain protein essential for segmentation and CNS development. Development 122, 3467–3475. 3046. Nardi, J.B. (1981). Epithelial invagination: adhesive properties of cells can govern position and directionality of epithelial folding. Differentiation 20, 97–103. 3047. Nardi, J.B. (1981). Induction of invagination in insect epithelium: paradigm for embryonic invagination. Science 214, 564–566. 3048. Nardi, J.B. (1994). Rearrangement of epithelial cell types in an insect wing monolayer is accompanied by differential expression of a cell surface protein. Dev. Dynamics 199, 315–325. 3049. Nardi, J.B. and Kafatos, F.C. (1976). Polarity and gradients in lepidopteran wing epidermis. I. Changes in graft polarity, form, and cell density accompanying transpositions and reorientations. J. Embryol. Exp. Morph. 36, 469–487. 3050. Nardi, J.B. and Kafatos, F.C. (1976). Polarity and gradients in lepidopteran wing epidermis. II. The differential adhesiveness model: gradient of a non-diffusible cell surface parameter. J. Embryol. Exp. Morph. 36, 489–512. 3051. Nardi, J.B. and Magee-Adams, S.M. (1986). Formation of scale spacing patterns in a moth wing. I. Epithelial feet may mediate cell rearrangement. Dev. Biol. 116, 265– 277. 3052. Nardi, J.B. and Norby, S.W. (1987). Evocation of venation pattern in the wing of a moth, Manduca sexta. J. Morphol. 193, 53–62. 3053. Nash, D. (1965). The expression of “Hairless” in Drosophila and the role of two closely linked modiﬁers of opposite effect. Genet. Res., Camb. 6, 175–189. 3054. Nash, D. (1970). The mutational basis for the “allelic” modiﬁer mutants, Enhancer and Suppressor of Hairless, of Drosophila melanogaster. Genetics 64, 471–479. 3055. Nash, W.G. (1976). Patterns of pigmentation color states regulated by the y locus in Drosophila melanogaster. Dev. Biol. 48, 336–343. 3056. Nash, W.G. and Yarkin, R.J. (1974). Genetic regulation and pattern formation: a study of the yellow locus in Drosophila melanogaster. Genet. Res., Camb. 24, 19–26. 3057. Nassar, N., Horn, G., Herrmann, C., Scherer, A., ˚ McCormick, F., and Wittinghofer, A. (1995). The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue. Nature 375, 554–560. 3058. Nassif, C., Daniel, A., Lengyel, J.A., and Hartenstein, V. (1998). The role of morphogenetic cell death during Drosophila embryonic head development. Dev. Biol. 197, 170–186. 3059. Natzle, J.E. (1993). Temporal regulation of Drosophila imaginal disc morphogenesis: a hierarchy of primary and secondary 20-hydroxyecdysone-responsive loci. Dev. Biol. 155, 516–532. 3060. Nauber, U., Pankratz, M.J., Kienlin, A., Seifert, E., Klemm, U., and J¨ackle, H. (1988). Abdominal segmentation of the

390

3061.

3062. 3063.

3064. 3065.

3066.

3067.

3068.

3069.

3070. 3071. 3072.

3073.

3074.

3075.

3076.

3077.

3078.

3079.

REFERENCES

Drosophila embryo requires a hormone receptor-like protein encoded by the gap gene knirps. Nature 336, 489– 492. Nayak, S.V. and Singh, R.N. (1983). Sensilla on the tarsal segments and mouthparts of adult Drosophila melanogaster Meigen (Diptera: Drosophilidae). Int. J. Insect Morphol. Embryol. 12, 273–291. Needham, J. (1933). On the dissociability of the fundamental processes in ontogenesis. Biol. Rev. 8, 180–223. Needham, J. (1936). “Order and Life.” Yale Univ. Pr., New Haven. (N.B. : Needham’s Fig. 11 predates Waddington’s 1940 idea of the “epigenetic landscape”; cf. Yoxen’s paper). Needham, J. (1959). “A History of Embryology.” 2nd ed. Cambridge Univ. Pr., Cambridge. Neel, B.G. and Tonks, N.K. (1997). Protein tyrosine phosphatases in signal transduction. Curr. Opin. Cell Biol. 9, 193–204. Neel, J.V. (1940). The interrelations of temperature, body size, and character expression in Drosophila melanogaster. Genetics 25, 225–250. Neel, J.V. (1940). The pattern of supernumerary macrochaetae in certain Drosophila mutants. Genetics 25, 251–277. Neel, J.V. (1941). Studies on the interaction of mutations affecting the chaetae of Drosophila melanogaster. I. The interaction of hairy, polychaetoid, and Hairy wing. Genetics 26, 52–68. Neel, J.V. (1943). Studies on the interaction of mutations affecting the chaetae of Drosophila melanogaster. II. The relation of character expression to size in ﬂies homozygous for polychaetoid, hairy, Hairy wing, and the combinations of these factors. Genetics 28, 49–68. Neel, J.V. (1983). Curt Stern: 1902–1981. Annu. Rev. Genet. 17, 1–10. Neel, J.V. (1987). Curt Stern: August 30, 1902–October 23, 1981. Biogr. Memoirs Natl. Acad. Sci. U. S. 56, 442–473. Neer, E.J., Schmidt, C.J., Nambudripad, R., and Smith, T.F. (1994). The ancient regulatory-protein family of WDrepeat proteins. Nature 371, 297–300. Nellen, D., Affolter, M., and Basler, K. (1994). Receptor serine/threonine kinases implicated in the control of Drosophila body pattern by decapentaplegic. Cell 78, 225– 237. Nellen, D., Burke, R., Struhl, G., and Basler, K. (1996). Direct and long-range action of a DPP morphogen gradient. Cell 85, 357–368. Nellesen, D.T., Lai, E.C., and Posakony, J.W. (1999). Discrete enhancer elements mediate selective responsiveness of Enhancer of split Complex genes to common transcriptional activators. Dev. Biol. 213, 33–53. Nelson, H.B., Heiman, R.G., Bolduc, C., Kovalick, G.E., Whitley, P., Stern, M., and Beckingham, K. (1997). Calmodulin point mutations affect Drosophila development and behavior. Genetics 147, 1783–1798. Nestoras, K., Lee, H., and Mohler, J. (1997). Role of knot (kn) in wing patterning in Drosophila. Genetics 147, 1203– 1212. Netter, S., Faucheux, M., Roignant, J.Y., Antoniewski, C., and Th´eodore, L. (2001). Developmental dynamics of Polycomb group proteins in vivo. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a97. Netter, S., Fauvarque, M.-O., del Corral, R.D., Dura, J.M., and Coen, D. (1998). white+ transgene insertions presenting a dorsal/ventral pattern deﬁne a single cluster of

3080.

3081.

3082.

3083.

3084.

3085.

3086.

3087. 3088.

3089.

3090.

3091.

3092.

3093.

3094.

3095.

3096.

3097.

homeobox genes that is silenced by the Polycomb-group proteins in Drosophila melanogaster. Genetics 149, 257– 275. Neubig, R.R. and Thomsen, W.J. (1989). How does a key ﬁt a ﬂexible lock? Structure and dynamics in receptor function. BioEssays 11, 136–141. Neufeld, T.P., de la Cruz, A.F.A., Johnston, L.A., and Edgar, B.A. (1998). Coordination of growth and cell division in the Drosophila wing. Cell 93, 1183–1193. Neufeld, T.P., Tang, A.H., and Rubin, G.M. (1998). A genetic screen to identify components of the sina signaling pathway in Drosophila eye development. Genetics 148, 277–286. Neuhold, L.A. and Wold, B. (1993). HLH forced dimers: Tethering MyoD to E47 generates a dominant positive myogenic factor insulated from negative regulation by Id. Cell 74, 1033–1042. Neuman-Silberberg, F.S., Schejter, E., Hoffmann, F.M., and Shilo, B.-Z. (1984). The Drosophila ras oncogenes: structure and nucleotide sequence. Cell 37, 1027–1033. Neuman-Silberberg, F.S. and Schupbach, T. (1994). Dorsoventral axis formation in Drosophila depends on the correct dosage of the gene gurken. Development 120, 2457–2463. ¨ Neuman-Silberberg, F.S. and Schupbach, T. (1993). The Drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGFα-like protein. Cell 75, 165–174. Neumann, C. and Cohen, S. (1997). Morphogens and pattern formation. BioEssays 19, 721–729. Neumann, C.J. and Cohen, S.M. (1996). Distinct mitogenic and cell fate speciﬁcation functions of wingless in different regions of the wing. Development 122, 1781–1789. Neumann, C.J. and Cohen, S.M. (1996). A hierarchy of cross-regulation involving Notch, wingless, vestigial and cut organizes the dorsal/ventral axis of the Drosophila wing. Development 122, 3477–3485. Neumann, C.J. and Cohen, S.M. (1996). Sternopleural is a regulatory mutation of wingless with both dominant and recessive effects on larval development of Drosophila melanogaster. Genetics 142, 1147–1155. Neumann, C.J. and Cohen, S.M. (1997). Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development 124, 871–880. Neumann, C.J. and Cohen, S.M. (1998). Boundary formation in Drosophila wing: Notch activity attenuated by the POU protein Nubbin. Science 281, 409–413. Neumann, C.J. and Nuesslein-Volhard, C. (2000). Patterning of the zebraﬁsh retina by a wave of sonic hedgehog activity. Science 289, 2137–2139. Newfeld, S.J., Chartoff, E.H., Graff, J.M., Melton, D.A., and Gelbart, W.M. (1996). Mothers against dpp encodes a conserved cytoplasmic protein required in DPP/TGF-β responsive cells. Development 122, 2099–2108. Newfeld, S.J., Mehra, A., Singer, M.A., Wrana, J.L., Attisano, L., and Gelbart, W.M. (1997). Mothers against dpp participates in a DPP/TGF-β responsive serine-threonine kinase signal transduction cascade. Development 124, 3167–3176. Newfeld, S.J., Padgett, R.W., Findley, S.D., Richter, B.G., Sanicola, M., de Cuevas, M., and Gelbart, W.M. (1997). Molecular evolution at the decapentaplegic locus in Drosophila. Genetics 145, 297–309. Newfeld, S.J., Su, M.A., and Wisotzkey, R.G. (2001). A

REFERENCES

3098.

3099.

3100. 3101.

3102.

3103.

3104.

3105.

3106.

3107.

3108.

3109.

3110.

3111.

3112.

screen for modiﬁers of decapentaplegic mutant phenotypes identiﬁes lilliputian, the only member of the FragileX/Burkitt’s lymphoma family of transcription factors in Drosophila melanogaster. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a164. [See also 2001 Genetics 157, 717–725.] Newfeld, S.J. and Takaesu, N.T. (1999). Local transposition of a hobo element within the decapentaplegic locus of Drosophila. Genetics 151, 177–187. Newfeld, S.J., Wisotzkey, R.G., and Kumar, S. (1999). Molecular evolution of a developmental pathway: phylogenetic analyses of Transforming Growth Factor-β family ligands, receptors and Smad signal transducers. Genetics 152, 783–795. Ng, H.H. and Bird, A. (2000). Histone deacetylases: silencers for hire. Trends Biochem. Sci. 25, 121–126. Ng, J., Hart, C.M., Morgan, K., and Simon, J.A. (2000). A Drosophila ESC-E(Z) protein complex is distinct from other Polycomb group complexes and contains covalently modiﬁed ESC. Mol. Cell. Biol. 20, 3069–3078. Ng, J., Li, R., Morgan, K., and Simon, J. (1997). Evolutionary conservation and predicted structure of the Drosophila extra sex combs repressor protein. Mol. Cell. Biol. 17, 6663–6672. Ng, M., Diaz-Benjumea, F.J., and Cohen, S.M. (1995). nubbin encodes a POU-domain protein required for proximal-distal patterning in the Drosophila wing. Development 121, 589–599. Ng, M., Diaz-Benjumea, F.J., Vincent, J.P., Wu, J., and Cohen, S.M. (1996). Speciﬁcation of the wing by localized expression of wingless protein. Nature 381, 316–318. Ng, M. and Yanofsky, M.F. (2001). Function and evolution of the plant MADS-box gene family. Nature Rev. Genet. 2, 186–195. Nguyen, D.N.T., Liu, Y., Litsky, M.L., and Reinke, R. (1997). The sidekick gene, a member of the immunoglobulin superfamily, is required for pattern formation in the Drosophila eye. Development 124, 3303–3312. Nguyen, D.N.T., Rohrbaugh, M., and Lai, Z.-C. (2000). The Drosophila homolog of Onecut homeodomain proteins is a neural-speciﬁc transcriptional activator with a potential role in regulating neural differentiation. Mechs. Dev. 97, 57–72. Nguyen, J.T., Turck, C.W., Cohen, F.E., Zuckermann, R.N., and Lim, W.A. (1998). Exploiting the basis of proline recognition by SH3 and WW domains: design of N-substituted inhibitors. Science 282, 2088–2092. Nguyen, M., Park, S., Marqu´es, G., and Arora, K. (1998). Interpretation of a BMP activity gradient in Drosophila embryos depends on synergistic signaling by two type I receptors, SAX and TKV. Cell 95, 495–506. Nguyen, M., Parker, L., and Arora, K. (2000). Identiﬁcation of maverick, a novel member of the TGF-β superfamily in Drosophila. Mechs. Dev. 95, 201–206. Nguyen, T., Jamal, J., Shimell, M.J., Arora, K., and O’Connor, M.B. (1994). Characterization of tolloidrelated-1: a BMP-1-like product that is required during larval and pupal stages of Drosophila development. Dev. Biol. 166, 569–586. Nibu, Y., Zhang, H., Bajor, E., Barolo, S., Small, S., and Levine, M. (1998). dCtBP mediates transcriptional repres¨ sion by Knirps, Kruppel and Snail in the Drosophila embryo. EMBO J. 17, 7009–7020. [See also 2001 EMBO J. 20, 2246–2253.]

391

3113. Nibu, Y., Zhang, H., and Levine, M. (1998). Interaction of short-range repressors with Drosophila CtBP in the embryo. Science 280, 101–104. [See also 2001 BioEssays 23, 683–690.] 3114. Nicholls, R.E. and Gelbart, W.M. (1998). Identiﬁcation of chromosomal regions involved in decapentaplegic function in Drosophila. Genetics 149, 203–215. 3115. Niehrs, C. and Pollet, N. (1999). Synexpression groups in eukaryotes. Nature 402, 483–487. 3116. Niessing, D., Dostatni, N., J¨ackle, H., and Rivera-Pomar, R. (1999). Sequence interval within the PEST motif of Bicoid is important for translational repression of caudal mRNA in the anterior region of the Drosophila embryo. EMBO J. 18, 1966–1973. 3117. Nigg, E.A. (1997). Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386, 779–787. 3118. Niimi, T., Seimiya, M., Kloter, U., Flister, S., and Gehring, W.J. (1999). Direct regulatory interaction of the eyeless protein with an eye-speciﬁc enhancer in the sine oculis gene during eye induction in Drosophila. Development 126, 2253–2260. 3119. Nijhout, H.F. (1991). “The Development and Evolution of Butterﬂy Wing Patterns.” Smithsonian Pr., Washington. ¨ 3120. Nilson, L.A. and Schupbach, T. (1999). EGF receptor signaling in Drosophila oogenesis. Curr. Topics Dev. Biol. 44, 203–243. 3121. Nilsson, D.-E. (1996). Eye ancestry: old genes for new eyes. Curr. Biol. 6, 39–42. 3122. Niwa, N., Inoue, Y., Nozawa, A., Saito, M., Misumi, Y., Ohuchi, H., Yoshioka, H., and Noji, S. (2000). Correlation of diversity of leg morphology in Gryllus bimaculatus (cricket) with divergence in dpp expression pattern during leg development. Development 127, 4373–4381. 3123. Nolan, G.P. and Baltimore, D. (1992). The inhibitory ankyrin and activator Rel proteins. Curr. Op. Gen. Dev. 2, 211–220. 3124. Noll, E., Medina, M., Hartley, D., Zhou, J., Perrimon, N., and Kosik, K.S. (2000). Presenilin affects Arm/β-catenin localization and function in Drosophila. Dev. Biol. 227, 450–464. 3125. Noll, M. (1993). Evolution and role of Pax genes. Curr. Opin. Gen. Dev. 3, 595–605. 3126. Noll, R., Sturtevant, M.A., Gollapudi, R.R., and Bier, E. (1994). New functions of the Drosophila rhomboid gene during embryonic and adult development are revealed by a novel genetic method, enhancer piracy. Development 120, 2329–2338. 3127. Nolo, R., Abbott, L.A., and Bellen, H.J. (2000). Senseless, a Zn ﬁnger transcription factor, is necessary and sufﬁcient for sensory organ development in Drosophila. Cell 102, 349–362. 3128. Nolo, R., Abbott, L.A., and Bellen, H.J. (2001). Drosophila Lyra mutations are gain-of-function mutations of senseless. Genetics 157, 307–315. 3129. Noordermeer, J., Klingensmith, J., and Nusse, R. (1995). Differential requirements for segment polarity genes in wingless signaling. Mechs. Dev. 51, 145–155. 3130. Noordermeer, J., Klingensmith, J., Perrimon, N., and Nusse, R. (1994). dishevelled and armadillo act in the Wingless signalling pathway in Drosophila. Nature 367, 80–83. 3131. Noramly, S. and Morgan, B.A. (1998). BMPs mediate lateral inhibition at successive stages in feather tract development. Development 125, 3775–3787.

392

3132. Norbeck, B.A. and Denburg, J.L. (1991). Pattern formation during insect leg segmentation: studies with a prepattern of a cell surface antigen. Roux’s Arch. Dev. Biol. 199, 476– 491. 3133. Nordenski¨old, E. (1936). “The History of Biology.” Tudor, New York. 3134. Nordstrom, W., Chen, P., Steller, H., and Abrams, J.M. (1996). Activation of the reaper gene during ectopic cell killing in Drosophila. Dev. Biol. 180, 213–226. 3135. North, G. (1986). Descartes and the fruitﬂy. Nature 322, 404–405. 3136. Norton, P.A., Hynes, R.O., and Rees, D.J.G. (1990). sevenless: seven found? Cell 61, 15–16. 3137. Nosseli, S. and Agn`es, F. (1999). Roles of the JNK signaling pathway in Drosophila morphogenesis. Curr. Opin. Gen. Dev. 9, 466–472. 3138. N¨othiger, R. (1964). Differenzierungsleistungen in Kombinaten, hergestellt aus Imaginalscheiben verschiedener Arten, Geschlechter und K¨orpersegmente von Drosophila. Roux’ Arch. Entw.-Mech. 155, 269–301. 3139. N¨othiger, R. (1972). The larval development of imaginal disks. In “The Biology of Imaginal Disks,” Results and Problems in Cell Differentiation, Vol. 5, (H. Ursprung and R. N¨othiger, eds.). Springer-Verlag: Berlin, pp. 1–34. 3140. N¨othiger, R. and Schubiger, G. (1966). Developmental behaviour of fragments of symmetrical and asymmetrical imaginal discs of Drosophila melanogaster (Diptera). J. Embryol. Exp. Morph. 16, 355–368. 3141. Nottebohm, E., Dambly-Chaudi`ere, C., and Ghysen, A. (1992). Connectivity of chemosensory neurons is controlled by the gene poxn in Drosophila. Nature 359, 829–832. 3142. Nottebohm, E., Usui, A., Therianos, S., Kimura, K.-i., Dambly-Chaudi`ere, C., and Ghysen, A. (1994). The gene poxn controls different steps of the formation of chemosensory organs in Drosophila. Neuron 12, 25–34. 3143. Noujdin, N.I. (1936). Genetic analysis of certain problems of the physiology of development of Drosophila melanogaster. Biol. Zh. (Russia) 5, 571–624. 3144. Nowak, M.A., Boerlijst, M.C., Cooke, J., and Maynard Smith, J. (1997). Evolution of genetic redundancy. Nature 388, 167–171. 3145. Nussbaumer, U., Halder, G., Groppe, J., Affolter, M., and Montagne, J. (2000). Expression of the blistered/DSRF gene is controlled by different morphogens during Drosophila trachea and wing development. Mechs. Dev. 96, 27–36. 3146. Nusse, R. (1997). A versatile transcriptional effector of Wingless signaling. Cell 89, 321–323. 3147. Nusse, R. (1999). Wnt targets: repression and activation. Trends Genet. 15, 1–3. 3148. Nusse, R., Brown, A., Papkoff, J., Scambler, P., Shackleford, G., McMahon, A., Moon, R., and Varums, H. (1991). A new nomenclature for int-1 and related genes: the Wnt gene family. Cell 64, 231. 3149. Nusse, R., Samos, C.H., Brink, M., Willert, K., Cadigan, K.M., Wodarz, A., Fish, M., and Rulifson, E. (1997). Cell culture and whole animal approaches to understanding signaling by Wnt proteins in Drosophila. Cold Spr. Harb. Symp. Quant. Biol. 62, 185–190. 3150. Nusse, R. and Varmus, H.E. (1992). Wnt genes. Cell 69, 1073–1087. ¨ 3151. Nusslein-Volhard, C. and Wieschaus, E. (1980). Mutations affecting segment number and polarity in Drosophila. Nature 287, 795–801.

REFERENCES

¨ 3152. Nusslein-Volhard, C., Wieschaus, E., and Kluding, H. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. I. Zygotic loci on the second chromosome. Roux’s Arch. Dev. Biol. 193, 267–282. 3153. Nye, J.S. (1997). Developmental signaling: Notch signals Kuz it’s cleaved. Curr. Biol. 7, R716–R720. 3154. O’Brochta, D.A. and Bryant, P.J. (1985). A zone of nonproliferating cells at a lineage restriction boundary in Drosophila. Nature 313, 138–141. 3155. O’Brochta, D.A. and Bryant, P.J. (1987). Distribution of S-phase cells during the regeneration of Drosophila imaginal wing discs. Dev. Biol. 119, 137–142. 3156. O’Connor, M.B., Binari, R., Perkins, L.A., and Bender, W. (1988). Alternative RNA products from the Ultrabithorax domain of the bithorax complex. EMBO J. 7, 435–445. 3157. O’Farrell, P.H., Desplan, C., DiNardo, S., Kassis, J.A., Kuner, J.M., Sher, E., Theis, J., and Wright, D. (1985). Embryonic pattern in Drosophila: the spatial distribution and sequence-speciﬁc DNA binding of engrailed protein. Cold Spr. Harb. Symp. Quant. Biol. 50, 235–242. 3158. O’Keefe, D.D. and Thomas, J.B. (2001). Drosophila wing development in the absence of dorsal identity. Development 128, 703–710. 3159. O’Keefe, D.D., Thor, S., and Thomas, J.B. (1998). Function and speciﬁcity of LIM domains in Drosophila nervous system and wing development. Development 125, 3915–3923. 3160. O’Neil, K.T., Hoess, R.H., and DeGrado, W.F. (1990). Design of DNA-binding peptides based on the leucine zipper motif. Science 249, 774–778. 3161. O’Neill, E.M., Ellis, M.C., Rubin, G.M., and Tjian, R. (1995). Functional domain analysis of glass, a zinc-ﬁngercontaining transcription factor in Drosophila. Proc. Natl. Acad. Sci. USA 92, 6557–6561. 3162. O’Neill, E.M., Rebay, I., Tjian, R., and Rubin, G.M. (1994). The activities of two Ets-related transcription factors required for Drosophila eye development are modulated by the Ras/MAPK pathway. Cell 78, 137–147. 3163. O’Shea, E.K., Klemm, J.D., Kim, P.S., and Alber, T. (1991). X-ray structure of the GCN4 leucine zipper, a twostranded, parallel coiled coil. Science 254, 539–544. 3164. O’Tousa, J.E., Baehr, W., Martin, R.L., Hirsh, J., Pak, W.L., and Applebury, M.L. (1985). The Drosophila ninaE gene encodes an opsin. Cell 40, 839–850. 3165. Oberlander, H. (1985). The imaginal discs. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology,” Vol. 2 (G.A. Kerkut and L.I. Gilbert, eds.). Pergamon Pr.: Oxford, pp. 151–182. 3166. Oda, H. and Tsukita, S. (1999). Dynamic features of adherens junctions during Drosophila embryonic epithelial morphogenesis revealed by a Dα-catenin-GFP fusion protein. Dev. Genes Evol. 209, 218–225. 3167. Oda, H., Tsukita, S., and Takeichi, M. (1998). Dynamic behavior of the cadherin-based cell-cell adhesion system during Drosophila gastrulation. Dev. Biol. 203, 435–450. 3168. Oda, H., Uemura, T., Harada, Y., Iwai, Y., and Takeichi, M. (1994). A Drosophila homolog of cadherin associated with Armadillo and essential for embryonic cell-cell adhesion. Dev. Biol. 165, 716–726. 3169. Oda, H., Uemura, T., Shiomi, K., Nagafuchi, A., Tsukita, S., and Takeichi, M. (1993). Identiﬁcation of a Drosophila homologue of α-catenin and its association with the armadillo protein. J. Cell Biol. 121, 1133–1140. 3170. Oeda, E., Oka, Y., Miyazono, K., and Kawabata, M. (1998). Interaction of Drosophila inhibitors of apoptosis with

REFERENCES

3171.

3172.

3173.

3174.

3175.

3176.

3177.

3178. 3179.

3180.

3181.

3182.

3183.

3184.

3185.

3186. 3187.

Thick veins, a type I serine/threonine kinase receptor for Decapentaplegic. J. Biol. Chem. 273#16, 9353–9356. Oellers, N., Dehio, M., and Knust, E. (1994). bHLH proteins encoded by the Enhancer of split complex of Drosophila negatively interfere with transcriptional activation mediated by proneural genes. Mol. Gen. Genet. 244, 465–473. Ogawa, E., Inuzuka, M., Maruyama, M., Satake, M., NaitoFujimoto, M., Ito, Y., and Shigesada, K. (1993). Molecular cloning and characterization of PEBP2β, the heterodimeric partner of a novel Drosophila runt-related DNA binding protein PEBP2α. Virology 194, 314–331. Ogawa, E., Maruyama, M., Kagoshima, H., Inuzuka, M., Lu, J., Satake, M., Shigesada, K., and Ito, Y. (1993). PEBP2/PEA2 represents a family of transcription factors homologous to the products of the Drosophila runt gene and the human AML1 gene. Proc. Natl. Acad. Sci. USA 90, 6859–6863. Ogryzko, V.V., Schiltz, R.L., Russanova, V., Howard, B.H., and Nakatani, Y. (1996). The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953– 959. Ohkuma, Y., Horikoshi, M., Roeder, R.G., and Desplan, C. (1990). Binding site-dependent direct activation and repression of in vitro transcription by Drosophila homeodomain proteins. Cell 61, 475–484. Ohlmeyer, J.T. and Kalderon, D. (1997). Dual pathways for induction of wingless expression by protein kinase A and Hedgehog in Drosophila embryos. Genes Dev. 11, 2250– 2258. Ohlmeyer, J.T. and Kalderon, D. (1998). Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator. Nature 396, 749–753. Ohno, S. (1970). “Evolution by Gene Duplication.” Springer-Verlag, Berlin. Ohsako, S., Hyer, J., Panganiban, G., Oliver, I., and Caudy, M. (1994). hairy function as a DNA-binding helix-loophelix repressor of Drosophila sensory organ formation. Genes Dev. 8, 2743–2755. Ohtsuki, S., Levine, M., and Cai, H.N. (1998). Different core promoters possess distinct regulatory activities in the Drosophila embryo. Genes Dev. 12, 547–556. Okabe, M. and Okano, H. (1997). Two-step induction of chordotonal organ precursors in Drosophila embryogenesis. Development 124, 1045–1053. Okano, H. (1995). Two major mechanisms regulating cellfate decisions in the developing nervous system. Develop. Growth Differ. 37, 619–629. Okano, H., Hayashi, S., Tanimura, T., Sawamoto, K., Yoshikawa, S., Watanabe, J., Iwasaki, M., Hirose, S., Mikoshiba, K., and Montell, C. (1992). Regulation of Drosophila neural development by a putative secreted protein. Differentiation 52, 1–11. Olayioye, M.A., Neve, R.M., Lane, H.A., and Hynes, N.E. (2000). The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J. 19, 3159–3167. Olivier, J.P., Raabe, T., Henkemeyer, M., Dickson, B., Mbamalu, G., Margolis, B., Schlessinger, J., Hafen, E., and Pawson, T. (1993). A Drosophila SH2-SH3 adaptor protein implicated in coupling the Sevenless tyrosine kinase to an activator of Ras Guanine Nucleotide Exchange, Sos. Cell 73, 179–191. Olson, E.N. (1990). MyoD family: a paradigm for development? Genes Dev. 4, 1454–1461. Oppenheim, R.W. (1981). Neuronal cell death and some

393

3188.

3189. 3190.

3191.

3192.

3193.

3194.

3195.

3196.

3197.

3198.

3199.

3200.

3201.

3202.

3203. 3204.

related regressive phenomena during neurogenesis: a selective historical review and progress report. In “Studies in Developmental Neurobiology. Essays in Honor of Viktor Hamburger,” (W.M. Cowan, ed.). Oxford Univ. Pr.: New York, pp. 74–133. Oppenheim, R.W. (1989). The neurotrophic theory and naturally occurring motoneuron death. Trends Neurosci. 12, 252–255. Oppenheim, R.W. (1991). Cell death during development of the nervous system. Annu. Rev. Neurosci. 14, 453–501. Oppenheimer, J.M. (1955). Problems, concepts and their history. In “Analysis of Development,” (B.H. Willier, P.A. Weiss, and V. Hamburger, eds.). W. B. Saunders: Philadephia, pp. 1–24. Orenic, T., Chidsey, J., and Holmgren, R. (1987). Cell and cubitus interruptus Dominant: two segment polarity genes on the fourth chromosome in Drosophila. Dev. Biol. 124, 50–56. Orenic, T.V. and Carroll, S.B. (1992). The cell biology of pattern formation during Drosophila development. Int. Rev. Cytol. 139, 121–155. Orenic, T.V., Held, L.I., Jr., Paddock, S.W., and Carroll, S.B. (1993). The spatial organization of epidermal structures: hairy establishes the geometrical pattern of Drosophila leg bristles by delimiting the domains of achaete expression. Development 118, 9–20. Orenic, T.V., Slusarski, D.C., Kroll, K.L., and Holmgren, R.A. (1990). Cloning and characterization of the segment polarity gene cubitus interruptus Dominant of Drosophila. Genes Dev. 4, 1053–1067. Orgogozo, V., Schweisguth, F., and Bella¨ıche, Y. (2001). Lineage, cell polarity and inscuteable function in the peripheral nervous system of the Drosophila embryo. Development 128, 631–643. Orlando, V., Jane, E.P., Chinwalla, V., Harte, P.J., and Paro, R. (1998). Binding of Trithorax and Polycomb proteins to the bithorax complex: dynamic changes during early Drosophila embryogenesis. EMBO J. 17, 5141–5150. Orlando, V. and Paro, R. (1995). Chromatin multiprotein complexes involved in the maintenance of transcription patterns. Curr. Opin. Gen. Dev. 5, 174–179. Oro, A.E., McKeown, M., and Evans, R.M. (1992). The Drosophila retinoid X receptor homolog ultraspiracle functions in both female reproduction and eye morphogenesis. Development 115, 449–462. Oro, A.E., Ong, E.S., Margolis, J.S., Posakony, J.W., McKeown, M., and Evans, R.M. (1988). The Drosophila gene knirps-related is a member of the steriod-receptor gene superfamily. Nature 336, 493–496. Oro, A.E. and Scott, M.P. (1998). Splitting hairs: dissecting roles of signaling systems in epidermal development. Cell 95, 575–578. Orr, H.T. (2001). Beyond the Qs in the polyglutamine diseases. Genes Dev. 15, 925–932. [See also 2001 Nature 413, 739–743.] Orsulic, S., Huber, O., Aberle, H., Arnold, S., and Kemler, R. (1999). E-cadherin binding prevents β-catenin nuclear localization and β-catenin/LEF-1-mediated transactivation. J. Cell Sci. 112, 1237–1245. Orsulic, S. and Peifer, M. (1996). Cell-cell signalling: Wingless lands at last. Curr. Biol. 6, 1363–1367. Orsulic, S. and Peifer, M. (1996). An in vivo structurefunction study of Armadillo, the β-catenin homologue, reveals both separate and overlapping regions of the

394

3205.

3206.

3207. 3208. 3209.

3210.

3211.

3212.

3213.

3214. 3215.

3216.

3217.

3218.

3219.

3220.

3221. 3222.

3223.

3224.

REFERENCES

protein required for cell adhesion and for Wingless signaling. J. Cell Biol. 134, 1283–1300. Oshiro, T., Yagami, T., Zhang, C., and Matsuzaki, F. (2000). Role of cortical tumour-suppressor proteins in asymmetric division of Drosophila neuroblast. Nature 408, 593– 596. Osten-Sacken, C.R. (1884). An essay of comparative chaetotaxy, or the arrangement of characteristic bristles of Diptera. Trans. Entomol. Soc. Lond. 1884 #4, 497– 517. Oster, G. (1988). Lateral inhibition models of developmental processes. Math. Biosci. 90, 265–286. Oster, G. and Weliky, M. (1991). Pattern and morphogenesis. Sems. Dev. Biol. 2, 139–150. Ota, K., Sakaguchi, M., von Heijne, G., Hamasaki, N., and Mihara, K. (1998). Forced transmembrane orientation of hydrophilic polypeptide segments in multispanning membrane proteins. Molec. Cell 2, 495–503. Ouweneel, W.J. (1969). Inﬂuence of environmental factors on the homoeotic effect of loboid-ophthalmoptera in Drosophila melanogaster. W. Roux’ Arch. 164, 15–36. Ouweneel, W.J. (1969). Morphology and development of loboid-ophthalmoptera, a homoeotic strain in Drosophila melanogaster. W. Roux’ Arch. 164, 1–14. Ouweneel, W.J. (1970). Developmental capacities of young and mature, wild-type and opht eye imaginal discs in Drosophila melanogaster. W. Roux’ Arch. 166, 76–88. Ouweneel, W.J. (1972). Determination, regulation, and positional information in insect development. Acta Biotheor. 21, 115–131. Ouweneel, W.J. (1976). Developmental genetics of homoeosis. Adv. Genet. 18, 179–248. Overduin, M., Rios, C.B., Mayer, B.J., Baltimore, D., and Cowburn, D. (1992). Three-dimensional solution structure of the src homology 2 domain of c-abl. Cell 70, 697– 704. Overton, J. (1967). The ﬁne structure of developing bristles in wild type and mutant Drosophila melanogaster. J. Morph. 122, 367–379. Owen, D.J. and Evans, P.R. (1998). A structural explanation for the recognition of tyrosine-based endocytotic signals. Science 282, 1327–1332. Owen, D.J., Wigge, P., Vallis, Y., Moore, J.D.A., Evans, P.R., and McMahon, H.T. (1998). Crystal structure of the amphiphysin-2 SH3 domain and its role in the prevention of dynamin ring formation. EMBO J. 17, 5273–5285. Pabo, C.O. and Sauer, R.T. (1992). Transcription factors: structural families and principles of DNA recognition. Annu. Rev. Biochem. 61, 1053–1095. Packert, G. and Kuhn, D.T. (1998). The tumoroushead-1 locus affects bristle number of the Drosophila melanogaster cuticle. Genetics 148, 743–752. Padgett, R.W. (1999). Intracellular signaling: ﬂeshing out the TGFβ pathway. Curr. Biol. 9, R408–R411. Padgett, R.W., Das, P., and Huang, H. (2001). Characterizing the Drosophila activin pathway. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a272. Padgett, R.W., Das, P., and Krishna, S. (1998). TGF-β signaling, Smads, and tumor suppressors. BioEssays 20, 382– 390. Padgett, R.W., St. Johnston, R.D., and Gelbart, W.M. (1987). A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-β family. Nature 325, 81–84.

3225. Pag`es, F. and Kerridge, S. (2000). Morphogen gradients: a question of time or concentration? Trends Genet. 16, 40–44. 3226. Pai, C.-Y., Kuo, T.-S., Jaw, T.J., Kurant, E., Chen, C.-T., Bessarab, D.A., Salzberg, A., and Sun, Y.H. (1998). The Homothorax homeoprotein activates the nuclear localization of another homeoprotein, Extradenticle, and suppresses eye development in Drosophila. Genes Dev. 12, 435–446. ¨ 3227. Pai, L.-M., Barcelo, G., and Schupbach, T. (2000). D-cbl, a negative regulator of the Egfr pathway, is required for dorsoventral patterning in Drosophila oogenesis. Cell 103, 51–61. 3228. Pai, L.-M., Orsulic, S., Bejsovec, A., and Peifer, M. (1997). Negative regulation of Armadillo, a Wingless effector in Drosophila. Development 124, 2255–2266. 3229. Pak, W.L., Conrad, S.K., Kremer, N.E., Larrivee, D.C., Schinz, R.H., and Wong, F. (1980). Photoreceptor function. In “Development and Neurobiology of Drosophila,” (O. Siddiqi, P. Babu, L.M. Hall, and J.C. Hall, eds.). Plenum: New York, pp. 331–346. 3230. Pak, W.L. and Grabowski, S.R. (1978). Physiology of the visual and ﬂight systems. In “The Genetics and Biology of Drosophila,” Vol. 2a (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 553–604. 3231. Pal-Bhadra, M., Bhadra, U., and Birchler, J.A. (1997). Cosuppression in Drosophila: gene silencing of Alcohol dehydrogenase by white-Adh transgenes is Polycomb dependent. Cell 90, 479–490. 3232. Palaparti, A., Baratz, A., and Stifani, S. (1997). The Groucho/Transducin-like Enhancer of split transcriptional repressors interact with the genetically deﬁned amino-terminal silencing domain of histone H3. J. Biol. Chem. 272 #42, 26604–26610. 3233. Palka, J. (1979). Mutants and mosaics: tools in insect developmental neurobiology. In “Aspects of Developmental Neurobiology,” Soc. Neurosci. Symp., Vol. 4 (J.A. Ferrendelli, ed.). Society for Neuroscience: Bethesda, MD, pp. 209–227. 3234. Palka, J., Schubiger, M., and Hart, H.S. (1981). The path of axons in Drosophila wings in relation to compartment boundaries. Nature 294, 447–449. 3235. Palka, J., Schubiger, M., and Schwaninger, H. (1990). Neurogenic and antineurogenic effects from modiﬁcations at the Notch locus. Development 109, 167–175. 3236. Palmeirim, I., Henrique, D., Ish-Horowicz, D., and Pourqui´e, O. (1997). Avian hairy gene expression identiﬁes a molecular clock linked to vertebrate segmentation and somitogenesis. Cell 91, 639–648. 3237. Palsson, A. and Gibson, G. (2000). Quantitative developmental genetic analysis reveals that the ancestral dipteran wing vein prepattern is conserved in Drosophila melanogaster. Dev. Genes Evol. 210, 617–622. 3238. Pan, D. and Rubin, G.M. (1995). cAMP-dependent protein kinase and hedgehog act antagonistically in regulating decapentaplegic transcription in Drosophila imaginal discs. Cell 80, 543–552. 3239. Pan, D. and Rubin, G.M. (1997). Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis. Cell 90, 271–280. 3240. Pan, D. and Rubin, G.M. (1998). Targeted expression of teashirt induces ectopic eyes in Drosophila. Proc. Natl. Acad. Sci. USA 95, 15508–15512.

REFERENCES

¨ 3241. Pandur, P. and Kuhl, M. (2001). An arrow for wingless to take-off. BioEssays 23, 207–210. 3242. Panganiban, G. (2000). Distal-less function during Drosophila appendage and sense organ development. Dev. Dynamics 218, 554–562. 3243. Panganiban, G., Nagy, L., and Carroll, S.B. (1994). The role of the Distal-less gene in the development and evolution of insect limbs. Curr. Biol. 4, 671–675. 3244. Panganiban, G.E.F., Rashka, K.E., Neitzel, M.D., and Hoffman, F.M. (1990). Biochemical characterization of the Drosophila dpp protein, a member of the Transforming Growth Factor β family of growth factors. Molec. Cell Biol. 10, 2669–2677. [See also 2001 Development 128, 2209– 2220.] 3245. Panin, V.M., Lei, L., Moloney, D.J., Shao, L., Haltiwanger, R.S., and Irvine, K.D. (2001). How does glycosylation by Fringe modulate Notch signaling? Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a153. 3246. Panin, V.M., Papayannopoulos, V., Wilson, R., and Irvine, K.D. (1997). Fringe modulates Notch-ligand interactions. Nature 387, 908–912. 3247. Pankratz, M.J. and J¨ackle, H. (1990). Making stripes in the Drosophila embryo. Trends Genet. 6, 287–292. 3248. Pankratz, M.J. and J¨ackle, H. (1993). Blastoderm segmentation. In “The Development of Drosophila melanogaster,” Vol. 1 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Lab. Pr.: Plainview, N. Y., pp. 467–516. 3249. Pannuti, A. and Lucchesi, J.C. (2000). Recycling to remodel: evolution of dosage-compensation complexes. Curr. Opin. Gen. Dev. 10, 644–650. 3250. Pantelouris, E.M. and Waddington, C.H. (1955). Regulation capacities of the wing- and haltere-discs of wild type and bithorax Drosophila. Roux’ Arch. Entw.-Mech. 147, 539–546. 3251. Paolella, D.N., Palmer, C.R., and Schepartz, A. (1994). DNA targets for certain bZIP proteins distinguished by an intrinsic bend. Science 264, 1130–1133. 3252. Papageorgiou, S. (1989). Cartesian or polar co-ordinates in pattern formation? J. Theor. Biol. 141, 281–283. 3253. Papageorgiou, S. (1996). Distalization in insects and amphibians. BioEssays 17, 1089–1090. 3254. Papaioannou, V.E. (1997). T-box family reunion. Trends Genet. 13, 212–213. 3255. Papaioannou, V.E. and Silver, L.M. (1998). The T-box gene family. BioEssays 20, 9–19. 3256. Papatsenko, D., Nazina, A., and Desplan, C. (2001). A conserved regulatory element present in all Drosophila rhodopsin genes mediates Pax6 functions and participates in the ﬁne-tuning of cell-speciﬁc expression. Mechs. Dev. 101, 143–153. 3257. Papatsenko, D., Sheng, G., and Desplan, C. (1997). A new rhodopsin in R8 photoreceptors of Drosophila: evidence for coordinate expression with Rh3 in R7 cells. Development 124, 1665–1673. 3258. Papayannopoulos, V., Tomlinson, A., Panin, V.M., Rauskolb, C., and Irvine, K.D. (1998). Dorsal-ventral signaling in the Drosophila eye. Science 281, 2031–2034. 3259. Papert, S. (1980). “Mindstorms: Children, Computers, and Powerful Ideas.” Basic Bks., New York. 3260. Papoulas, O., Beek, S.J., Moseley, S.L., McCallum, C.M., Sarte, M., Shearn, A., and Tamkun, J.W. (1998). The Drosophila trithorax group proteins BRM, ASH1 and ASH2 are subunits of distinct protein complexes. Development 125, 3955–3966.

395

3261. Paricio, N., Feiguin, F., Boutros, M., Eaton, S., and Mlodzik, M. (1999). The Drosophila STE20-like kinase Misshapen is required downstream of the Frizzled receptor in planar polarity signaling. EMBO J. 18, 4669–4678. 3262. Park, J.M., Gim, B.S., Kim, J.M., Yoon, J.H., Kim, H.-S., Kang, J.-G., and Kim, Y.-J. (2001). Drosophila Mediator complex is broadly utilized by diverse gene-speciﬁc transcription factors at different types of core promoters. Molec. Cell. Biol. 21, 2312–2323. 3263. Park, M., Yaich, L.E., and Bodmer, R. (1998). Mesodermal cell fate decisions in Drosophila are under the control of the lineage genes numb, Notch, and sanpodo. Mechs. Dev. 75, 117–126. 3264. Park, W.-J., Liu, J., and Adler, P.N. (1994). The frizzled gene of Drosophila encodes a membrane protein with an odd number of transmembrane domains. Mechs. Dev. 45, 127– 137. 3265. Parkhurst, S.M. (1998). Groucho: making its Marx as a transcriptional co-repressor. Trends Genet. 14, 130–132. 3266. Parkhurst, S.M., Bopp, D., and Ish-Horowicz, D. (1990). X:A ratio, the primary sex-determining signal in Drosophila, is transduced by helix-loop-helix proteins. Cell 63, 1179–1191. 3267. Parkhurst, S.M. and Ish-Horowicz, D. (1991). Misregulating segmentation gene expression in Drosophila. Development 111, 1121–1135. 3268. Parkhurst, S.M. and Ish-Horowicz, D. (1992). Common denominators for sex. Curr. Biol. 2, 629–631. 3269. Parkin, N.T., Kitajewski, J., and Varmus, H.E. (1993). Activity of Wnt-1 as a transmembrane protein. Genes Dev. 7, 2181–2193. 3270. Parks, A.L., Huppert, S.S., and Muskavitch, M.A.T. (1997). The dynamics of neurogenic signalling underlying bristle development in Drosophila melanogaster. Mechs. Dev. 63, 61–74. 3271. Parks, A.L., Klueg, K.M., Stout, J.R., and Muskavitch, M.A.T. (2000). Ligand endocytosis drives receptor dissociation and activation in the Notch pathway. Development 127, 1373–1385. 3272. Parks, A.L. and Muskavitch, M.A.T. (1993). Delta function is required for bristle organ determination and morphogenesis in Drosophila. Dev. Biol. 157, 484–496. 3273. Parks, A.L., Turner, R., and Muskavitch, M.A.T. (1995). Relationships between complex Delta expression and the speciﬁcation of retinal cell fates during Drosophila eye development. Mechs. Dev. 50, 201–216. 3274. Parks, H.B. (1936). Cleavage patterns in Drosophila and mosaic formation. Ann. Ent. Soc. Am. 29, 350–392. 3275. Paro, R., Strutt, H., and Cavalli, G. (1998). Heritable chromatin states induced by the Polycomb and trithorax group genes. In “Epigenetics,” Novartis Foundation Symposium, Vol. 214 (D.J. Chadwick and G. Cardew, eds.). Wiley & Sons: New York, pp. 51–66. [See also 2001 BioEssays 23, 561–562.] 3276. Paro, R. and Zink, B. (1992). The Polycomb gene is differentially regulated during oogenesis and embryogenesis of Drosophila melanogaster. Mechs. Dev. 40, 37–46. 3277. Parody, T.R. and Muskavitch, M.A.T. (1993). The pleiotropic function of Delta during postembryonic development of Drosophila melanogaster. Genetics 135, 527–539. 3278. Paroush, Z., Finley, R.L., Jr., Kidd, T., Wainwright, S.M., Ingham, P.W., Brent, R., and Ish-Horowicz, D. (1994). Groucho is required for Drosophila neurogenesis, segmentation, and sex determination and interacts directly with Hairyrelated bHLH proteins. Cell 79, 805–815.

396

3279. Parvin, J.D. and Young, R.A. (1998). Regulatory targets in the RNA polymerase II holoenzyme. Curr. Opin. Gen. Dev. 8, 565–570. 3280. Pascal, S.M., Singer, A.U., Gish, G., Yamazaki, T., Shoelson, S.E., Pawson, T., Kay, L.E., and Forman-Kay, J.D. (1994). Nuclear magnetic resonance structure of an SH2 domain of phospholipase C-γ1 complexed with a high afﬁnity binding peptide. Cell 77, 461–472. 3281. Passner, J.M., Ryoo, H.D., Shen, L., Mann, R.S., and Aggarwal, A.K. (1999). Structure of a DNA-bound UltrabithoraxExtradenticle homeodomain complex. Nature 397, 714– 719. 3282. Patel, K., Makarenkova, H., and Jung, H.-S. (1999). The role of long range, local and direct signalling molecules during chick feather bud development involving the BMPs, Follistatin and the Eph receptor tyrosine kinase Eph-A4. Mechs. Dev. 86, 51–62. 3283. Paterson, J. and O’Hare, K. (1991). Structure and transcription of the singed locus of Drosophila melanogaster. Genetics 129, 1073–1084. 3284. Patikoglou, G. and Burley, S.K. (1997). Eukaryotic transcription factor-DNA complexes. Annu. Rev. Biophys. Biomol. Struct. 26, 289–325. [See also 2001 Molec. Cell 8, 937–946.] 3285. Pattatucci, A.M. and Kaufman, T.C. (1991). The homeotic gene Sex combs reduced of Drosophila melanogaster is differentially regulated in the embryonic and imaginal stages of development. Genetics 129, 443–461. 3286. Pattatucci, A.M., Otteson, D.C., and Kaufman, T.C. (1991). A functional and structural analysis of the Sex combs reduced locus of Drosophila melanogaster. Genetics 129, 423–441. 3287. Pattee, H.H. (1969). How does a molecule become a message? In “Communication in Development,” Symp. Soc. Dev. Biol., Vol. 28 (A. Lang, ed.). Acad. Pr.: New York, pp. 1–16. 3288. Pattee, H.H. (1972). The nature of hierarchical controls in living matter. In “Foundations of Mathematical Biology,” Vol. 1 (R. Rosen, ed.). Acad. Pr.: New York, pp. 1–22. 3289. Patthy, L. (1991). Modular exchange principles in proteins. Curr. Opinion Struct. Biol. 1, 351–361. 3290. Paulus, H.F. (1979). Eye structure and the monophyly of the Arthropoda. In “Arthropod Phylogeny,” (A.P. Gupta, ed.). Van Nostrand Reinhold: New York, pp. 299–383. 3291. Paumard-Rigal, S., Zider, A., Vaudin, P., and Silber, J. (1998). Speciﬁc interactions between vestigial and scalloped are required to promote wing tissue proliferation in Drosophila melanogaster. Dev. Genes Evol. 208, 440–446. 3292. Pavletich, N.P. and Pabo, C.O. (1993). Crystal structure of a ﬁve-ﬁnger GLI-DNA complex: new perspectives on zinc ﬁngers. Science 261, 1701–1707. 3293. Pawley, J. (2000). The 39 steps: a cautionary tale of quantitative 3-D ﬂuorescence microscopy. BioTechniques 28, 884–888. 3294. Pawson, T. (1993). Signal transduction–a conserved pathway from the membrane to the nucleus. Dev. Genet. 14, 333–338. 3295. Pawson, T. (1995). Protein modules and signalling networks. Nature 373, 573–580. 3296. Pawson, T. and Gish, G.D. (1992). SH2 and SH3 domains: from structure to function. Cell 71, 359–362. 3297. Pawson, T. and Nash, P. (2000). Protein-protein interactions deﬁne speciﬁcity in signal transduction. Genes Dev. 14, 1027–1047.

REFERENCES

3298. Pawson, T. and Schlessinger, J. (1993). SH2 and SH3 domains. Curr. Biol. 3, 434–442. 3299. Pawson, T. and Scott, J.D. (1997). Signaling through scaffold, anchoring, and adaptor proteins. Science 278, 2075– 2080. 3300. Pazman, C., Mayes, C.A., Fanto, M., Haynes, S.R., and Mlodzik, M. (2000). Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in Rasand Rho-mediated signaling. Development 127, 1715– 1725. 3301. Pearson, M.J. (1974). Polyteny and the functional signiﬁcance of the polytene cell cycle. J. Cell Sci. 15, 457–479. 3302. Pederson, T. (2001). Protein mobility within the nucleus– what are the right moves? Cell 104, 635–638. 3303. Peel, D.J., Johnson, S.A., and Milner, M.J. (1990). The ultrastructure of imaginal disc cells in primary cultures and during cell aggregation in continuous cell lines. Tissue & Cell 22, 749–758. 3304. Peifer, M. (1993). Cancer, catenins, and cuticle pattern: a complex connection. Science 262, 1667–1668. 3305. Peifer, M. (1994). The two faces of Hedgehog. Science 266, 1492–1493. 3306. Peifer, M. (1995). Cell adhesion and signal transduction: the Armadillo connection. Trends Cell Biol. 5, 224–229. 3307. Peifer, M. (1996). Regulating cell proliferation: as easy as APC. Science 272, 974–975. 3308. Peifer, M. (1998). Birds of a feather ﬂock together. Nature 395, 324–325. 3309. Peifer, M. (1999). Neither straight nor narrow. Nature 400, 213–215. 3310. Peifer, M. and Bejsovec, A. (1992). Knowing your neighbors: cell interactions determine intrasegmental patterning in Drosophila. Trends Genet. 8, 243–249. 3311. Peifer, M. and Bender, W. (1986). The anterobithorax and bithorax mutations of the bithorax complex. EMBO J. 5, 2293–2303. 3312. Peifer, M., Berg, S., and Reynolds, A.B. (1994). A repeating amino acid motif shared by proteins with diverse cellular roles. Cell 76, 789–791. 3313. Peifer, M., Karch, F., and Bender, W. (1987). The bithorax complex: control of segmental identity. Genes Dev. 1, 891– 898. 3314. Peifer, M., Orsulic, S., Sweeton, D., and Wieschaus, E. (1993). A role for the Drosophila segment polarity gene armadillo in cell adhesion and cytoskeletal integrity during oogenesis. Development 118, 1191–1207. 3315. Peifer, M., Pai, L.-M., and Casey, M. (1994). Phosphorylation of the Drosophila adherens junction protein armadillo: roles for wingless signal and zeste-white 3 kinase. Dev. Biol. 166, 543–556. 3316. Peifer, M. and Polakis, P. (2000). Wnt signaling in oncogenesis and embryogenesis–a look outside the nucleus. Science 287, 1606–1609. 3317. Peifer, M., Rauskolb, C., Williams, M., Riggleman, B., and Wieschaus, E. (1991). The segment polarity gene armadillo interacts with the wingless signalling pathway in both embryonic and adult pattern formation. Development 111, 1029–1043. 3318. Peifer, M., Sweeton, D., Casey, M., and Wieschaus, E. (1994). wingless signal and Zeste-white 3 kinase trigger opposing changes in the intracellular distribution of Armadillo. Development 120, 369–380. 3319. Peifer, M. and Wieschaus, E. (1990). Mutations in the Drosophila gene extradenticle affect the way speciﬁc

REFERENCES

3320.

3321.

3322.

3323.

3324.

3325.

3326.

3327. 3328.

3329.

3330.

3331.

3332.

3333.

3334.

3335.

homeo domain proteins regulate segmental identity. Genes Dev. 4, 1209–1223. Peifer, M. and Wieschaus, E. (1990). The segment polarity gene armadillo encodes a functionally modular protein that is the Drosophila homolog of human plakoglobin. Cell 63, 1167–1178. Pelaz, S., Urqu´ıa, N., and Morata, G. (1993). Normal and ectopic domains of the homeotic gene Sex combs reduced of Drosophila. Development 117, 917–923. Pellegrini, L., Burke, D.F., von Delft, F., Mulloy, B., and Blundell, T.L. (2000). Crystal structure of ﬁbroblast growth factor receptor ectodomain bound to ligand and heparin. Nature 407, 1029–1034. Pellegrini, L., Tan, S., and Richmond, T.J. (1995). Structure of serum response factor core bound to DNA. Nature 376, 490–498. Peltenburg, L.T.C. and Murre, C. (1996). Engrailed and Hox homeodomain proteins contain a related Pbx interaction motif that recognizes a common structure present in Pbx. EMBO J. 15, 3385–3393. Peltenburg, L.T.C. and Murre, C. (1997). Speciﬁc residues in the Pbx homeodomain differentially modulate the DNA-binding activity of Hox and Engrailed proteins. Development 124, 1089–1098. Peng, C.-Y., Manning, L., Albertson, R., and Doe, C.Q. (2000). The tumour-suppressor genes lgl and dlg regulate basal protein targeting in Drosophila neuroblasts. Nature 408, 596–600. Pennisi, E. (1997). Multiple clocks keep time in fruit ﬂy tissues. Science 278, 1560–1561. Penton, A., Chen, Y., Staehling-Hampton, K., Wrana, J.L., Attisano, L., Szidonya, J., Cassill, J.A., Massagu´e, J., and Hoffmann, F.M. (1994). Identiﬁcation of two bone morphogenetic protein type 1 receptors in Drosophila and evidence that Brk25D is a decapentaplegic receptor. Cell 78, 239–250. Penton, A. and Hoffmann, M. (1996). Decapentaplegic restricts the domain of wingless during Drosophila limb patterning. Nature 382, 162–165. Penton, A., Selleck, S.B., and Hoffmann, F.M. (1997). Regulation of cell cycle synchronization by decapentaplegic during Drosophila eye development. Science 275, 203– 206. Pepling, M.E. and Gergen, J.P. (1995). Conservation and function of the transcriptional regulatory protein Runt. Proc. Natl. Acad. Sci. USA 92, 9087–9091. Percival-Smith, A. and Hayden, D.J. (1998). Analysis in Drosophila melanogaster of the interaction between Sex combs reduced and Extradenticle activity in the determination of tarsus and arista identity. Genetics 150, 189– 198. Percival-Smith, A., Weber, J., Gilfoyle, E., and Wilson, P. (1997). Genetic characterization of the role of the two HOX proteins, Proboscipedia and Sex Combs Reduced, in determination of adult antennal, tarsal, maxillary palp and proboscis identities in Drosophila melanogaster. Development 124, 5049–5062. Perissi, V., Dasen, J.S., Kurokawa, R., Wang, Z., Korzus, E., Rose, D.W., Glass, C.K., and Rosenfeld, M.G. (1999). Factorspeciﬁc modulation of CREB-binding protein acetyltransferase activity. Proc. Natl. Acad. Sci. USA 96, 3652–3657. Periz, G. and Fortini, M.E. (1999). Ca2+ -ATPase function is required for intracellular trafﬁcking of the Notch receptor in Drosophila. EMBO J. 18, 5983–5993.

397

3336. Perkins, L.A., Johnson, M.R., Melnick, M.B., and Perrimon, N. (1996). The nonreceptor protein tyrosine phosphatase Corkscrew functions in multiple receptor tyrosine kinase pathways in Drosophila. Dev. Biol. 180, 63–81. 3337. Perkins, L.A., Larsen, I., and Perrimon, N. (1992). corkscrew encodes a putative protein tyrosine phosphatase that functions to transduce the terminal signal from the receptor tyrosine kinase torso. Cell 70, 225–236. 3338. Perler, F.B. (1998). Protein splicing of inteins and Hedgehog autoproteolysis: structure, function, and evolution. Cell 92, 1–4. 3339. Perlman, D. and Halvorson, H.O. (1983). A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J. Mol. Biol. 167, 391–409. 3340. Perrimon, N. (1993). The torso receptor protein-tyrosine kinase signaling pathway: an endless story. Cell 74, 219– 222. 3341. Perrimon, N. (1994). The genetic basis of patterned baldness in Drosophila. Cell 76, 781–784. 3342. Perrimon, N. (1994). Signalling pathways initiated by receptor protein tyrosine kinases in Drosophila. Curr. Opin. Cell Biol. 6, 260–266. 3343. Perrimon, N. (1995). Hedgehog and beyond. Cell 80, 517– 520. 3344. Perrimon, N. (1996). Serpentine proteins slither into the Wingless and Hedgehog ﬁelds. Cell 86, 513–516. 3345. Perrimon, N. and Bernﬁeld, M. (2000). Speciﬁcities of heparan sulphate proteoglycans in developmental processes. Nature 404, 725–728. 3346. Perrimon, N. and Duffy, J.B. (1998). Sending all the right signals. Nature 396, 18–19. 3347. Perrimon, N. and McMahon, A.P. (1999). Negative feedback mechanisms and their roles during pattern formation. Cell 97, 13–16. 3348. Perrimon, N. and Perkins, L.A. (1997). There must be 50 ways to rule the signal: the case of the Drosophila EGF receptor. Cell 89, 13–16. 3349. Perrimon, N. and Smouse, D. (1989). Multiple functions of a Drosophila homeotic gene, zeste-white 3, during segmentation and neurogenesis. Dev. Biol. 135, 287–305. 3350. Perry, M.M. (1968). Further studies on the development of the eye of Drosophila melanogaster. I. The ommatidia. J. Morph. 124, 227–248. 3351. Perry, M.M. (1968). Further studies on the development of the eye of Drosophila melanogaster. II. The interommatidial bristles. J. Morph. 124, 249–262. 3352. Peruski, L.F., Jr., Wadzinski, B.E., and Johnson, G.L. (1993). Analysis of the multiplicity, structure, and function of protein serine/threonine phosphatases. Adv. Prot. Phosphatases 7, 9–30. 3353. Perutz, M.F., Johnson, T., Suzuki, M., and Finch, J.T. (1994). Glutamine repeats as polar zippers: Their possible role in inherited neurodegenerative diseases. Proc. Natl. Acad. Sci. USA 91, 5355–5358. 3354. Perutz, M.F., Staden, R., Moens, L., and De Baere, I. (1993). Polar zippers. Curr. Biol. 3, 249–253. 3355. Petcherski, A.G. and Kimble, J. (2000). Mastermind is a putative activator for Notch. Curr. Biol. 10, R471–R473. 3356. Petersen, N.S., Lankenau, D.-H., Mitchell, H.K., Young, P., and Corces, V.G. (1994). forked proteins are components of ﬁber bundles present in developing bristles of Drosophila melanogaster. Genetics 136, 173–182. 3357. Pettigrew, J.B. (1908). “Design in Nature.” Vol. 1. Longmans, Green, & Co., London.

398

3358. Pettmann, B. and Henderson, C.E. (1998). Neuronal cell death. Neuron 20, 633–647. 3359. Peverali, F.A., Isaksson, A., Papavassiliou, A.G., Plastina, P., Staszewski, L.M., Mlodzik, M., and Bohmann, D. (1996). Phosphorylation of Drosophila Jun by the MAP kinase Rolled regulates photoreceptor differentiation. EMBO J. 15, 3943–3950. 3360. Pevny, L.H. and Lovell-Badge, R. (1997). Sox genes ﬁnd their feet. Curr. Opin. Genet. Dev. 7, 338–344. 3361. Peyer, B. (1947). An early description of Drosophila. J. Heredity 38, 194–199. 3362. Peyer, B. and Hadorn, E. (1965). Zum Manifestationsmuster der Mutante “multiple wing hairs” (mwh) von Drosophila melanogaster. Arch. Klaus-Stift. Vererb.Forsch. 40, 19–26. 3363. Pfeiffer, S., Alexandre, C., Calleja, M., and Vincent, J.-P. (2000). The progeny of wingless-expressing cells deliver the signal at a distance in Drosophila embryos. Curr. Biol. 10, 321–324. 3364. Pfeiffer, S. and Vincent, J.-P. (1999). Signalling at a distance: Transport of Wingless in the embryonic epidermis of Drosophila. Sems. Cell Dev. Biol. 10, 303–309. 3365. Pﬂugfelder, G.O., Roth, H., and Poeck, B. (1992). A homology domain shared between Drosophila optomotorblind and mouse Brachyury is involved in DNA binding. Biochem. Biophys. Res. Comm. 186, 918–925. 3366. Pﬂugfelder, G.O., Roth, H., Poeck, B., Kerscher, S., Schwarz, H., Jonschker, B., and Heisenberg, M. (1992). The lethal(1)optomotor-blind gene of Drosophila melanogaster is a major organizer of optic lobe development: isolation and characterization of the gene. Proc. Natl. Acad. Sci. USA 89, 1199–1203. 3367. Pﬂugfelder, G.O., Schwarz, H., Roth, H., Poeck, B., Sigl, A., Kerscher, S., Jonschker, B., Pak, W.L., and Heisenberg, M. (1990). Genetic and molecular characterization of the optomotor-blind gene locus in Drosophila melanogaster. Genetics 126, 91–104. 3368. Pham, A., Therond, P., Alves, G., Tournier, F.B., Busson, D., Lamour-Isnard, C., Bouchon, B.L., Pr´eat, T., and Tricoire, H. (1995). The Suppressor of fused gene encodes a novel PEST protein involved in Drosophila segment polarity establishment. Genetics 140, 587–598. 3369. Phillips, J.P. and Forrest, H.S. (1980). Ommochromes and pteridines. In “The Genetics and Biology of Drosophila,” Vol. 2d (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 541–623. 3370. Phillips, M.D. and Shearn, A. (1990). Mutations in polycombeotic, a Drosophila Polycomb-group gene, cause a wide range of maternal and zygotic phenotypes. Genetics 125, 91–101. 3371. Phillips, R.G. (2001). A novel gene, skinhead, regulates vestigial expression and may be involved in Wingless signal transduction. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a200. 3372. Phillips, R.G., Roberts, I.J.H., Ingham, P.W., and Whittle, J.R.S. (1990). The Drosophila segment polarity gene patched is involved in a position-signalling mechanism in imaginal discs. Development 110, 105–114. 3373. Phillips, R.G., Warner, N.L., and Whittle, J.R.S. (1999). Wingless signaling leads to an asymmetric response to Decapentaplegic-dependent signaling during sense organ patterning on the notum of Drosophila melanogaster. Dev. Biol. 207, 150–162. 3374. Phillips, R.G. and Whittle, J.R.S. (1993). wingless expres-

REFERENCES

3375.

3376.

3377.

3378.

3379. 3380.

3381.

3382.

3383. 3384.

3385.

3386.

3387.

3388.

3389. 3390.

3391.

sion mediates determination of peripheral nervous system elements in late stages of Drosophila wing disc development. Development 118, 427–438. Phillips, S.E.V. (1994). Built by association: structure and function of helix-loop-helix DNA-binding proteins. Curr. Biol. 2, 1–4. Phillis, R.W., Bramlage, A.T., Wotus, C., Whittaker, A., Gramates, L.S., Seppala, D., Farahanchi, F., Caruccio, P., and Murphey, R.K. (1993). Isolation of mutations affecting neural circuitry required for grooming behavior in Drosophila melanogaster. Genetics 133, 581–592. Phippen, T.M., Sweigart, A.L., Moniwa, M., Krumm, A., Davie, J.R., and Parkhurst, S.M. (2000). Drosophila C-terminal binding protein functions as a contextdependent transcriptional co-factor and interferes with both Mad and Groucho transcriptional repression. J. Biol. Chem. 275 #48, 37628–37637. Pi, H., Wu, H.-J., and Chien, C.-T. (2001). A dual function of phyllopod in Drosophila external sensory organ development: cell fate speciﬁcation of sensory organ precursor and its progeny. Development 128, 2699–2710. Piatigorsky, J. and Wistow, G.J. (1989). Enzyme/crystallins: gene sharing as an evolutionary strategy. Cell 57, 197–199. Pichaud, F. and Casares, F. (2000). homothorax and iroquois-C genes are required for the establishment of territories within the developing eye disc. Mechs. Dev. 96, 15–25. Pichaud, F. and Desplan, C. (2001). A new visualization approach for identifying mutations that affect differentiation and organization of the Drosophila ommatidia. Development 128, 815–826. Pichaud, F., Treisman, J., and Desplan, C. (2001). Reinventing a common strategy for patterning the eye. Cell 105, 9–12. Pick, L. (1998). Segmentation: painting stripes from ﬂies to vertebrates. Dev. Genet. 23, 1–10. Pickett, F.B. and Meeks-Wagner, D.R. (1995). Seeing double: appreciating genetic redundancy. The Plant Cell 7, 1347–1356. Pickup, A.T. and Banerjee, U. (1999). The role of Star in the production of an activated ligand for the EGF receptor signaling pathway. Development 205, 254–259. [See also 2001 Curr. Biol. 12, R21–R23, 2002 Genes Dev. 16, 222–234, and 2001 Mechs. Dev. 107, 13–23.] ¨ Piepenburg, O., Vorbruggen, G., and J¨ackle, H. (2000). Drosophila segment borders result from unilateral repression of Hedgehog activity by Wingless signaling. Molec. Cell 6, 203–209. Pignoni, F., Hu, B., Zavitz, K.H., Xiao, J., Garrity, P.A., and Zipursky, S.L. (1997). The eye-speciﬁcation proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development. Cell 91, 881–891. Pignoni, F. and Zipursky, S.L. (1997). Induction of Drosophila eye development by Decapentaplegic. Development 124, 271–278. Pilpel, Y. and Lancet, D. (1999). Good reception in fruitﬂy antennae. Nature 398, 285–287. Pimentel, A.C. and Venkatesh, T.R. (2001). The retina aberrant in pattern (rap) gene encodes the Drosophila Fizzy related protein (Fzr) and regulates cell cycle and pattern formation in the developing eye. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a65. Pinsonneault, J., Florence, B., Vaessin, H., and McGinnis, W. (1997). A model for extradenticle function as a switch

REFERENCES

3392.

3393.

3394. 3395.

3396. 3397.

3398.

3399. 3400. 3401.

3402.

3403.

3404.

3405.

3406. 3407.

3408.

3409.

3410.

that changes HOX proteins from repressors to activators. EMBO J. 16, 2032–2042. Pinto, M. and Lobe, C.G. (1996). Products of the grg (groucho-related gene) family can dimerize through the amino-terminal Q domain. J. Biol. Chem. 271 #51, 33026– 33031. Piper, D.E., Batchelor, A.H., Chang, C.-P., Cleary, M.L., and Wolberger, C. (1999). Structure of a HoxB1-Pbx1 heterodimer bound to DNA: role of the hexapeptide and a fourth homeodomain helix in complex formation. Cell 96, 587–597. Pirrotta, V. (1995). Chromatin complexes regulating gene expression in Drosophila. Curr. Opin. Gen. Dev. 5, 466–472. Pirrotta, V. (1997). Chromatin-silencing mechanisms in Drosophila maintain patterns of gene expression. Trends Genet. 13, 314–318. Pirrotta, V. (1998). Polycombing the genome: PcG, trxG, and chromatin silencing. Cell 93, 333–336. Pirrotta, V., Chan, C.S., McCabe, D., and Qian, S. (1995). Distinct parasegmental and imaginal enhancers and the establishment of the expression pattern of the Ubx gene. Genetics 141, 1439–1450. Piternick, L.K., ed. (1980). “Richard Goldschmidt: Controversial Geneticist and Creative Biologist. A Critical Review of His Contributions.” Birkh¨auser, Basel. Placzek, M. and Skaer, H. (1999). Airway patterning: a paradigm for restricted signaling. Curr. Biol. 9, R506–R510. Plasterk, R.H.A. and Ketting, R.F. (2000). The silence of the genes. Curr. Opin. Gen. Dev. 10, 562–567. Plautz, J.D., Kaneko, M., Hall, J.C., and Kay, S.A. (1997). Independent photoreceptive circadian clocks throughout Drosophila. Science 278, 1632–1635. Plaza, S., Prince, F., Jaeger, J., Kloter, U., Flister, S., Benassayag, C., Cribbs, D., and Gehring, W.J. (2001). Molecular basis for the inhibition of Drosophila eye development by Antennapedia. EMBO J. 20, 802–811. [See also 2001 Development 128, 3307–3319.] Plotnikov, A.N., Hubbard, S.R., Schlessinger, J., and Mohammadi, M. (2000). Crystal structures of two FGF-FGFR complexes reveal the determinants of ligand-receptor speciﬁcity. Cell 101, 413–424. Plotnikov, A.N., Schlessinger, J., Hubbard, S.R., and Mohammadi, M. (1999). Structural basis for FGF receptor dimerization and activation. Cell 98, 641–650. Plunkett, C.R. (1926). The interaction of genetic and environmental factors in development. J. Exp. Zool. 46, 181– 244. Podos, S.D. and Ferguson, E.L. (1999). Morphogen gradients: new insights from DPP. Trends Genet. 15, 396–402. Poeck, B., Fischer, S., Gunning, D., Zipursky, S.L., and Salecker, I. (2001). Glial cells mediate target layer selection of retinal axons in the developing visual system of Drosophila. Neuron 29, 99–113. [See also 2001 Neuron 30, 437–450.] Poeck, B., Hofbauer, A., and Pﬂugfelder, G.O. (1993). Expression of the Drosophila optomotor-blind gene transcript in neuronal and glial cells of the developing nervous system. Development 117, 1017–1029. Pokutta, S. and Weis, W.I. (2000). Structure of the dimerization and β-catenin-binding region of α-catenin. Molec. Cell 5, 533–543. Polaczyk, P.J., Gasperini, R., and Gibson, G. (1998). Naturally occurring genetic variation affects Drosophila photoreceptor determination. Dev. Genes Evol. 207, 462–470.

399

3411. Polakis, P. (2000). Wnt signaling and cancer. Genes Dev. 14, 1837–1851. 3412. Pollard, T.D., Blanchoin, L., and Mullins, R.D. (2000). Molecular mechanisms controlling actin ﬁlament dynamics in nonmuscle cells. Annu. Rev. Biophys. Biomol. Struct. 29, 545–576. 3413. Pollard, V.W., Michael, W.M., Nakielny, S., Siomi, M.C., Wang, F., and Dreyfuss, G. (1996). A novel receptormediated nuclear protein import pathway. Cell 86, 985– 994. 3414. Pollock, J.A. and Benzer, S. (1988). Transcript localization of four opsin genes in the three visual organs of Drosophila: RH2 is ocellus speciﬁc. Nature 333, 779–782. 3415. Polyak, S. (1957). “The Vertebrate Visual System.” Univ. Chicago Pr., Chicago. 3416. Polymenis, M. and Schmidt, E.V. (1999). Coordination of cell growth with cell division. Curr. Opin. Gen. Dev. 9, 76– 80. 3417. Ponting, C.P. (1995). AF-6/cno: neither a kinesin nor a myosin, but a bit of both. Trends Biochem. Sci. 20, 265– 266. 3418. Ponting, C.P. and Benjamin, D.R. (1996). A novel family of Ras-binding domains. Trends Biochem. Sci. 21, 422–425. 3419. Ponting, C.P., Phillips, C., Davies, K.E., and Blake, D.J. (1997). PDZ domains: targeting signalling molecules to sub-membranous sites. BioEssays 19, 469–479. 3420. Poodry, C.A. (1975). A temporal pattern in the development of sensory bristles in Drosophila. W. Roux’s Arch. 178, 203–213. 3421. Poodry, C.A. (1980). Epidermis: morphology and development. In “The Genetics and Biology of Drosophila,” Vol. 2d (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 443–497. 3422. Poodry, C.A. (1980). Imaginal discs: morphology and development. In “The Genetics and Biology of Drosophila,” Vol. 2d (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 407–441. 3423. Poodry, C.A. (1990). shibire, a neurogenic mutant of Drosophila. Dev. Biol. 138, 464–472. 3424. Poodry, C.A., Bryant, P.J., and Schneiderman, H.A. (1971). The mechanism of pattern reconstruction by dissociated imaginal discs of Drosophila melanogaster. Dev. Biol. 26, 464–477. 3425. Poodry, C.A., Hall, L., and Suzuki, D.T. (1973). Developmental properties of shibirets : a pleiotropic mutation affecting larval and adult locomotion and development. Dev. Biol. 32, 373–386. 3426. Poodry, C.A. and Schneiderman, H.A. (1970). The ultrastructure of the developing leg of Drosophila melanogaster. W. Roux’ Arch. 166, 1–44. 3427. Poodry, C.A. and Schneiderman, H.A. (1976). Pattern formation in Drosophila melanogaster : the effects of mutations on polarity in the developing leg. W. Roux’s Arch. 180, 175–188. 3428. Poodry, C.A. and Woods, D.F. (1990). Control of the developmental timer for Drosophila pupariation. Roux’s Arch. Dev. Biol. 199, 219–227. 3429. Poole, S.J., Kauvar, L.M., Drees, B., and Kornberg, T. (1985). The engrailed locus of Drosophila: structural analysis of an embryonic transcript. Cell 40, 37–43. 3430. Poortinga, G., Watanabe, M., and Parkhurst, S.M. (1998). Drosophila CtBP: a Hairy-interacting protein required for embryonic segmentation and Hairy-mediated transcriptional repression. EMBO J. 17, 2067–2078.

400

3431. Popadic, A., Abzhanov, A., Rusch, D., and Kaufman, T.C. (1998). Understanding the genetic basis of morphological evolution: the role of homeotic genes in the diversiﬁcation of the arthropod bauplan. Int. J. Dev. Biol. 42, 453–461. 3432. Porter, J.A., Ekker, S.C., Park, W.-J., von Kessler, D.P., Young, K.E., Chen, C.-H., Ma, Y., Woods, A.S., Cotter, R.J., Koonin, E.V., and Beachy, P.A. (1996). Hedgehog patterning activity: role of a lipophilic modiﬁcation mediated by the carboxyterminal autoprocessing domain. Cell 86, 21–34. 3433. Porter, J.A., von Kessler, D.P., Ekker, S.C., Young, K.E., Lee, J.J., Moses, K., and Beachy, P.A. (1995). The product of hedgehog autoproteolytic cleavage active in local and long-range signalling. Nature 374, 363–366. 3434. Porter, J.A., Young, K.E., and Beachy, P.A. (1996). Cholesterol modiﬁcation of Hedgehog signaling proteins in animal development. Science 274, 255–259. 3435. Portin, P. (1975). Allelic negative complementation at the Abruptex locus of Drosophila melanogaster. Genetics 81, 121–133. 3436. Portin, P. (1981). The antimorphic mode of action of lethal Abruptex alleles of the Notch locus in Drosophila melanogaster. Hereditas 95, 247–251. 3437. Posakony, J.W. (1994). Nature versus nurture: asymmetric cell divisions in Drosophila bristle development. Cell 76, 415–418. 3438. Posakony, L.G., Raftery, L.A., and Gelbart, W.M. (1991). Wing formation in Drosophila melanogaster requires decapentaplegic gene function along the anterior-posterior compartment boundary. Mechs. Dev. 33, 69–82. 3439. Postlethwait, J.H. (1974). Development of the temperature-sensitive homoeotic mutant ophthalmoptera of Drosophila melanogaster. Dev. Biol. 36, 212–217. 3440. Postlethwait, J.H. (1975). Pattern formation in the wing and haltere imaginal discs after irradiation of Drosophila melanogaster ﬁrst instar larvae. W. Roux’ Arch. 178, 29–50. 3441. Postlethwait, J.H. (1978). Clonal analysis of Drosophila cuticular patterns. In “The Genetics and Biology of Drosophila,” Vol. 2c (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 359–441. 3442. Postlethwait, J.H. (1978). Development of cuticular patterns in the legs of a cell lethal mutant of Drosophila melanogaster. W. Roux’s Arch. 185, 37–57. 3443. Postlethwait, J.H. and Girton, J. (1974). Development of antennal-leg homoeotic mutants in Drosophila melanogaster. Genetics 76, 767–774. 3444. Postlethwait, J.H. and Girton, J.R. (1974). Development in genetic mosaics of aristapedia, a homoeotic mutant of Drosophila melanogaster. Genetics 76, 767–774. 3445. Postlethwait, J.H. and Schneiderman, H.A. (1969). A clonal analysis of determination in Antennapedia, a homoeotic mutant of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 64, 176–183. 3446. Postlethwait, J.H. and Schneiderman, H.A. (1971). A clonal analysis of development in Drosophila melanogaster : morphogenesis, determination, and growth in the wildtype antenna. Dev. Biol. 24, 477–519. 3447. Postlethwait, J.H. and Schneiderman, H.A. (1971). Pattern formation and determination in the antenna of the homoeotic mutant Antennapedia of Drosophila melanogaster. Dev. Biol. 25, 606–640. 3448. Postlethwait, J.H. and Schneiderman, H.A. (1973). Developmental genetics of Drosophila imaginal discs. Annu. Rev. Genetics 7, 381–433. 3449. Postlethwait, J.H. and Schneiderman, H.A. (1973). Pattern

REFERENCES

3450.

3451. 3452.

3453.

3454. 3455.

3456.

3457. 3458.

3459.

3460.

3461.

3462. 3463.

3464.

3465.

3466.

3467.

formation in imaginal discs of Drosophila melanogaster after irradiation of embryos and young larvae. Dev. Biol. 32, 345–360. Potter, C.J., Huang, H., and Xu, T. (2001). Drosophila Tsc1 functions with dTsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell 105, 357–368. [See also 2002 BioEssays 24, 54–64; 2001 Cell 105, 345–355; 2001 Genes Dev. 15, 1383–1392.] Pourqui´e, O. (1999). Notch around the clock. Curr. Opin. Gen. Dev. 9, 559–565. [See also 2001 J.Anat. 199, 169–175.] Poux, S., Kostic, C., and Pirrotta, V. (1996). Hunchbackindependent silencing of late Ubx enhancers by a Polycomb Group Response Element. EMBO J. 15, 4713–4722. Poux, S., McCabe, D., and Pirrotta, V. (2001). Recruitment of components of Polycomb Group chromatin complexes in Drosophila. Development 128, 75–85. [See also 2001 Nature 412, 655–660.] Pouyss´egur, J. (2000). An arresting start for MAPK. Science 290, 1515–1518. Powe, A.C., Jr., Strathdee, D., Cutforth, T., D’Souza-Correia, T., Gaines, P., Thackeray, J., Carlson, J., and Gaul, U. (1999). In vivo functional analysis of Drosophila Gap1: involvement of Ca2+ and IP4 regulation. Mechs. Dev. 81, 89–101. Powell, P.A., Wesley, C., Spencer, S., and Cagan, R.L. (2001). Scabrous complexes with Notch to mediate boundary formation. Nature 409, 626–630. Pradel, J. and White, R.A.H. (1998). From selectors to realizators. Int. J. Dev. Biol. 42, 417–421. ´ A. (1999). The haplolethal Prado, A., Canal, I., and Ferrus, region at the 16F gene cluster of Drosophila melanogaster: structure and function. Genetics 151, 163–175. Pr´eat, T. (1992). Characterization of Suppressor of fused, a complete suppressor of the fused segment gene of Drosophila melanogaster. Genetics 132, 725–736. Pr´eat, T., Th´erond, P., Lamour-Isnard, C., LimbourgBouchon, B., Tricoire, H., Erk, I., Mariol, M.-C., and Busson, D. (1990). A putative serine/threonine protein kinase encoded by the segment-polarity fused gene of Drosophila. Nature 347, 87–89. Pr´eat, T., Th´erond, P., Limbourg-Bouchon, B., Pham, A., Tricoire, H., Busson, D., and Lamour-Isnard, C. (1993). Segmental polarity in Drosophila melanogaster : genetic dissection of fused in a Suppressor of fused background reveals interaction with costal-2. Genetics 135, 1047–1062. Prelich, G. (1999). Suppression mechanisms: themes from variations. Trends Genet. 15, 261–266. Price, B.D., Chang, Z., Smith, R., Bockheim, S., and Laughon, A. (1993). The Drosophila neuralized gene encodes a C3 HC4 zinc ﬁnger. EMBO J. 12, 2411–2418. ¨ Price, J.V., Clifford, R.J., and Schupbach, T. (1989). The maternal ventralizing locus torpedo is allelic to faint little ball, an embryonic lethal, and encodes the Drosophila EGF receptor homolog. Cell 56, 1085–1092. Price, J.V., Savenye, E.D., Lum, D., and Breitkreutz, A. (1997). Dominant enhancers of Egfr in Drosophila melanogaster: genetic links between the Notch and Egfr signaling pathways. Genetics 147, 1139–1153. Price, M.A. and Kalderon, D. (1999). Proteolysis of Cubitus interruptus in Drosophila requires phosphorylation by Protein Kinase A. Development 126, 4331–4339. Price, M.D. and Lai, Z.-C. (1999). The yan gene is highly conserved in Drosophila and its expression suggests a complex role throughout development. Dev. Genes Evol. 209, 207–217.

REFERENCES

3468. Price, M.H., McCartney, B.M., Rayner, J., Bejsovec, A., and Peifer, M. (2001). Characterization of new alleles of Drosophila APC2, a cytoskeletally associated protein which negatively regulates the Wingless pathway. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a172. 3469. Primakoff, P. and Myles, D.G. (2000). The ADAM gene family. Trends Genet. 16, 83–87. 3470. Prober, D.A. and Edgar, B.A. (2000). Ras1 promotes cellular growth in the Drosophila wing. Cell 100, 435–446. 3471. Prokopenko, S.N., He, Y., Lu, Y., and Bellen, H.J. (2000). Mutations affecting the development of the peripheral nervous system in Drosophila: a molecular screen for novel proteins. Genetics 156, 1691–1715. 3472. Prout, M., Damania, Z., Soong, J., Fristrom, D., and Fristrom, J. (1997). Autosomal mutations affecting adhesion between wing surfaces in Drosophila melanogaster. Genetics 146, 275–285. 3473. Ptashne, M. and Gann, A. (1998). Imposing speciﬁcity by localization: mechanism and evolvabilty. Curr. Biol. 8, R812–R822. 3474. Pueyo, J.I., Galindo, M.I., Bishop, S.A., and Couso, J.P. (2000). Proximal-distal leg development in Drosophila requires the apterous gene and the Lim1 homologue dlim1. Development 127, 5391–5402. 3475. Pulido, D., Campuzano, S., Koda, T., Modolell, J., and Barbacid, M. (1992). Dtrk, a Drosophila gene related to the trk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule. EMBO J. 11, 391–404. 3476. Punzo, C., Seimiya, M., Schnupf, P., Gehring, W.J., and Plaza, S. (2001). The sine oculis enhancer is regulated by two Drosophila Pax-6 genes. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a116. 3477. Purugganan, M.D. (1998). The molecular evolution of development. BioEssays 20, 700–711. 3478. Purves, D. (1988). “Body and Brain: A Trophic Theory of Neural Connections.” Harvard, Cambridge, Mass. 3479. Qi, H., Rand, M.D., Wu, X., Sestan, N., Wang, W., Rakic, P., Xu, T., and Artavanis-Tsakonas, S. (1999). Processing of the Notch ligand Delta by the metalloprotease Kuzbanian. Science 283, 91–94. [But cf. 2002 Genes Dev. 16, 209–221.] 3480. Qian, S., Capovilla, M., and Pirrotta, V. (1991). The bx region enhancer, a distant cis-control element of the Drosophila Ubx gene and its regulation by hunchback and other segmentation genes. EMBO J. 10, 1415–1425. 3481. Qian, S., Capovilla, M., and Pirrotta, V. (1993). Molecular mechanisms of pattern formation by the BRE enhancer of the Ubx gene. EMBO J. 12, 3865–3877. ¨ 3482. Qian, Y.Q., Billeter, M., Otting, G., Muller, M., Gehring, W.J., ¨ and Wuthrich, K. (1989). The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution: comparison with prokaryotic repressors. Cell 59, 573–580. 3483. Qin, Y., Luo, Z.-Q., Smyth, A.J., Gao, P., von Bodman, S.B., and Farrand, S.K. (2000). Quorum-sensing signal binding results in dimerization of TraR and its release from membranes into the cytoplasm. EMBO J. 19, 5212–5221. ¨ 3484. Queenan, A.M., Ghabrial, A., and Schupbach, T. (1997). Ectopic activation of torpedo/Egfr, a Drosophila receptor tyrosine kinase, dorsalizes both the eggshell and the embryo. Development 124, 3871–3880. 3485. Quilliam, L.A., Khosravi-Far, R., Huff, S.Y., and Der, C.J. (1995). Guanine nucleotide exchange factors: activators of the Ras superfamily of proteins. BioEssays 17, 395–404. 3486. Quiring, R., Walldorf, U., Kloter, U., and Gehring, W.J.

401

3487.

3488.

3489.

3490.

3491.

3492. 3493.

3494.

3495. 3496.

3497.

3498.

3499.

3500.

3501.

3502.

3503.

(1994). Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans. Science 265, 785–789. Quirk, J., van den Heuvel, M., Henrique, D., Marigo, V., Jones, T.A., Tabin, C., and Ingham, P.W. (1997). The smoothened gene and Hedgehog signal transduction in Drosophila and vertebrate development. Cold Spr. Harb. Symp. Quant. Biol. 62, 217–226. Raabe, T., Olivier, J.P., Dickson, B., Liu, X., Gish, G.D., Pawson, T., and Hafen, E. (1995). Biochemical and genetic analysis of the Drk SH2/SH3 adaptor protein of Drosophila. EMBO J. 14, 2509–2518. Raabe, T., Riesgo-Escovar, J., Liu, X., Bausenwein, B.S., Deak, P., Mar¨oy, P., and Hafen, E. (1996). DOS, a novel pleckstrin homology domain-containing protein required for signal transduction between Sevenless and Ras1 in Drosophila. Cell 85, 911–920. Rabinow, L. and Birchler, J.A. (1990). Interactions of vestigial and scabrous with the Notch locus of Drosophila melanogaster. Genetics 125, 41–50. Radke, K., Baek, K.-H., and Ambrosio, L. (1997). Characterization of maternal and zygotic D-raf proteins: dominant negative effects on Torso signal transduction. Genetics 145, 163–171. Raff, M.C. (1996). Size control: the regulation of cell numbers in animal development. Cell 86, 173–175. Raff, M.C., Barres, B.A., Burne, J.F., Coles, H.S., Ishizaki, Y., and Jacobson, M.D. (1993). Programmed cell death and the control of cell survival: lessons from the nervous system. Science 262, 695–700. Raff, R.A. (1996). “The Shape of Life: Genes, Development, and the Evolution of Animal Form.” Univ. Chicago Pr., Chicago. Raff, R.A. (2000). Evo-devo: the evolution of a new discipline. Nature Rev. Gen. 1, 74–79. Raff, R.A. and Sly, B.J. (2000). Modularity and dissociation in the evolution of gene expression territories in development. Evol. Dev. 2, 102–113. Raftery, L.A., Sanicola, M., Blackman, R.K., and Gelbart, W.M. (1991). The relationship of decapentaplegic and engrailed expression in Drosophila imaginal disks: do these genes mark the anterior-posterior compartment boundary? Development 113, 27–33. Raftery, L.A. and Sutherland, D.J. (1999). TGF-β family signal transduction in Drosophila development: from Mad to Smads. Dev. Biol. 210, 251–268. Raftery, L.A., Twombly, V., Wharton, K., and Gelbart, W.M. (1995). Genetic screens to identify elements of the decapentaplegic signaling pathway in Drosophila. Genetics 139, 241–254. Raj, L., Vivekanand, P., Das, T.K., Badam, E., Fernandes, M., Finley, R.L., Jr., Brent, R., Appel, L.F., Hanes, S.D., and Weir, M. (2000). Targeted localized degradation of Paired protein in Drosophila development. Curr. Biol. 10, 1265– 1272. Ralston, A. and Blair, S.S. (2001). BMP-like signaling and the crossveinless 2-dependent pathway of crossvein development. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a201. Ramaekers, G., Usui, K., Usui-Ishihara, A., Ramaekers, A., Ledent, V., Ghysen, A., and Dambly-Chaudi`ere, C. (1997). Lineage and fate in Drosophila: role of the gene tramtrack in sense organ development. Dev. Genes Evol. 207, 97–106. Ramain, P., Heitzler, P., Haenlin, M., and Simpson, P. (1993).

402

3504.

3505.

3506.

3507.

3508.

3509.

3510.

3511.

3512.

3513.

3514.

3515. 3516.

3517. 3518.

3519.

3520.

3521.

REFERENCES

pannier, a negative regulator of achaete and scute in Drosophila, encodes a zinc ﬁnger protein with homology to the vertebrate transcription factor GATA-1. Development 119, 1277–1291. Ramain, P., Khechumian, R., Khechumian, K., Arbogast, N., Ackermann, C., and Heitzler, P. (2000). Interactions between Chip and the Achaete/Scute-Daughterless heterodimers are required for Pannier-driven proneural patterning. Molec. Cell 6, 781–790. Ramel, M.-C. and Katz, F.N. (2001). A novel role for enabled in leg segmentation: expression and genetic analysis. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a201. Ram´ırez-Weber, F.-A., Casso, D.J., Aza-Blanc, P., Tabata, T., and Kornberg, T.B. (2000). Hedgehog signal transduction in the posterior compartment of the Drosophila wing imaginal disc. Molec. Cell 6, 479–485. Ram´ırez-Weber, F.-A. and Kornberg, T.B. (1999). Cytonemes: cellular processes that project to the principal signaling center in Drosophila imaginal discs. Cell 97, 599– 607. Ram´ırez-Weber, F.-A. and Kornberg, T.B. (2000). Signaling reaches to new dimensions in Drosophila imaginal discs. Cell 103, 189–192. Ramos, R.G.P., Igloi, G.L., Lichte, B., Baumann, U., Maier, D., Schneider, T., Brandst¨atter, J.H., Fr¨ohlich, A., and Fischbach, K.-F. (1993). The irregular chiasm Croughest locus of Drosophila, which affects axonal projections and programmed cell death, encodes a novel immunoglobulin-like protein. Genes Dev. 7, 2533–2547. Randall, L.L. and Hardy, S.J.S. (1989). Unity of function in the absence of consensus in sequence: role of leader peptides in export. Science 243, 1156–1159. Randazzo, F.M., Seeger, M.A., Huss, C.A., Sweeney, M.A., Cecil, J.K., and Kaufman, T.C. (1993). Structural changes in the Antennapedia complex of Drosophila pseudoobscura. Genetics 134, 319–330. Randsholt, N.B., Maschat, F., and Santamaria, P. (2000). polyhomeotic controls engrailed expression and the hedgehog signaling pathway in imaginal discs. Mechs. Dev. 95, 89–99. Ranganathan, R. and Ross, E.M. (1997). PDZ domain proteins: scaffolds for signaling complexes. Curr. Biol. 7, R770–R773. Rangarajan, R., Gong, Q., and Gaul, U. (1999). Migration and function of glia in the developing Drosophila eye. Development 126, 3285–3292. Ransom, R. (1975). Computer analysis of division patterns in the Drosophila head disc. J. Theor. Biol. 53, 445–462. Ransom, R. (1982). Eye and head development. In “A Handbook of Drosophila Development,” (R. Ransom, ed.). Elsevier: Amsterdam, pp. 123–152. Ransom, R. (1982). “A Handbook of Drosophila Development.” Elsevier, Amsterdam. Ransom, R. and Matela, R.J. (1984). Computer modelling of cell division during development using a topological approach. J. Embryol. Exp. Morph. 83 (Suppl.), 233–259. Rao, Y., Bodmer, R., Jan, L.Y., and Jan, Y.N. (1992). The big brain gene of Drosophila functions to control the number of neuronal precursors in the peripheral nervous system. Development 116, 31–40. Rao, Y., Jan, L.Y., and Jan, Y.N. (1990). Similarity of the product of the Drosophila neurogenic gene big brain to transmembrane channel proteins. Nature 345, 163–167. Rao, Z., Handford, P., Mayhew, M., Knott, V., Brownlee,

3522. 3523. 3524.

3525.

3526.

3527.

3528.

3529.

3530.

3531.

3532.

3533.

3534.

3535.

3536.

3537.

3538. 3539.

G.G., and Stuart, D. (1995). The structure of a Ca2+ -binding epidermal growth factor-like domain: its role in proteinprotein interactions. Cell 82, 131–141. Rasmuson, M. (1952). Variation in bristle number of Drosophila melanogaster. Acta Zool. 33, 277–307. Rauskolb, C. (2001). The establishment of segmentation in the Drosophila leg. Development 128, 4511–4521. Rauskolb, C., Correia, T., and Irvine, K.D. (1999). Fringedependent separation of dorsal and ventral cells in the Drosophila wing. Nature 401, 476–480. Rauskolb, C. and Irvine, K.D. (1999). Notch-mediated segmentation and growth control of the Drosophila leg. Dev. Biol. 210, 339–350. Rauskolb, C., Peifer, M., and Wieschaus, E. (1993). extradenticle, a regulator of homeotic gene activity, is a homolog of the homeobox-containing human protooncogene pbx1. Cell 74, 1101–1112. Rauskolb, C., Smith, K.M., Peifer, M., and Wieschaus, E. (1995). extradenticle determines segmental identities throughout Drosophila development. Development 121, 3663–3673. Rauskolb, C. and Wieschaus, E. (1994). Coordinate regulation of downstream genes by extradenticle and the homeotic selector proteins. EMBO J. 13, 3561–3569. Ray, K., Hartenstein, V., and Rodrigues, V. (1993). Development of the taste bristles on the labellum of Drosophila melanogaster. Dev. Biol. 155, 26–37. Ray, K. and Rodrigues, V. (1994). The function of the proneural genes achaete and scute in the spatio-temporal patterning of the adult labellar bristles of Drosophila melanogaster. Roux’s Arch. Dev. Biol. 203, 340–350. Ray, K. and Rodrigues, V. (1995). Cellular events during development of the olfactory sense organs in Drosophila melanogaster. Dev. Biol. 167, 426–438. ¨ Ray, R.P., Arora, K., Nusslein-Volhard, C., and Gelbart, W.M. (1991). The control of cell fate along the dorsalventral axis of the Drosophila embryo. Development 113, 35–54. Ray, W.J., Yao, M., Mumm, J., Schroeter, E.H., Saftig, P., Wolfe, M., Selkoe, D.J., Kopan, R., and Goate, A.M. (1999). Cell surface Presenilin-1 participates in the γ-secretaselike proteolysis of Notch. J. Biol. Chem. 274 #51, 36801– 36807. Ray, W.J., Yao, M., Nowotny, P., Mumm, J., Zhang, W., Wu, J.Y., Kopan, R., and Goate, A.M. (1999). Evidence for a physical interaction between presenilin and Notch. Proc. Natl. Acad. Sci. USA 96, 3263–3268. Raz, E. and Shilo, B.-Z. (1992). Dissection of the faint little ball (ﬂb) phenotype: determination of the development of the Drosophila central nervous system by early interactions in the ectoderm. Development 114, 113–123. Raz, E. and Shilo, B.-Z. (1993). Establishment of ventral cell fates in the Drosophila embryonic ectoderm requires DER, the EGF receptor homolog. Genes Dev. 7, 1937–1948. Read, D. and Manley, J.L. (1992). Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc ﬁnger proteins with distinct DNA binding speciﬁcities. EMBO J. 11, 1035–1044. [See also 2001 Proc. Natl. Acad. Sci. USA 98, 9724–9729.] Ready, D.F. (1989). A multifaceted approach to neural development. Trends Neurosci. 12, 102–110. Ready, D.F., Hanson, T.E., and Benzer, S. (1976). Development of the Drosophila retina, a neurocrystalline lattice. Dev. Biol. 53, 217–240.

REFERENCES

3540. Ready, D.F., Tomlinson, A., and Lebovitz, R.M. (1986). Building an ommatidium: geometry and genes. In “Development of Order in the Visual System,” (S.R. Hilfer and J.B. Shefﬁeld, eds.). Springer-Verlag: Berlin, pp. 97–125. 3541. Rebagliati, M. (1989). An RNA recognition motif in the bicoid protein. Cell 58, 231–232. 3542. Rebay, I., Chen, F., Hsiao, F., Kolodziej, P.A., Kuang, B.H., Laverty, T., Suh, C., Voas, M., Williams, A., and Rubin, G.M. (2000). A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identiﬁes split ends, a new RNA recognition motif-containing protein. Genetics 154, 695–712. 3543. Rebay, I., Fehon, R.G., and Artavanis-Tsakonas, S. (1993). Speciﬁc truncations of Drosophila Notch deﬁne dominant activated and dominant negative forms of the receptor. Cell 74, 319–329. 3544. Rebay, I., Fleming, R.J., Fehon, R.G., Cherbas, L., Cherbas, P., and Artavanis-Tsakonas, S. (1991). Speciﬁc EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor. Cell 67, 687–699. 3545. Rebay, I. and Rubin, G.M. (1995). Yan functions as a general inhibitor of differentiation and is negatively regulated by activation of the Ras1/MAPK pathway. Cell 81, 857–866. 3546. Rebecchi, M.J. and Scarlata, S. (1998). Pleckstrin homology domains: a common fold with diverse functions. Annu. Rev. Biophys. Biomol. Struct. 27, 503–528. 3547. Rechsteiner, M. and Rogers, S.W. (1996). PEST sequences and regulation by proteolysis. Trends Biochem. Sci. 21, 267–271. 3548. Reddy, G.V., Gupta, B., Ray, K., and Rodrigues, V. (1997). Development of the Drosophila olfactory sense organs utilizes cell-cell interactions as well as lineage. Development 124, 703–712. 3549. Reddy, G.V. and Rodrigues, V. (1999). A glial cell arises from an additional division within the mechanosensory lineage during development of the microchaete on the Drosophila notum. Development 126, 4617–4622. 3550. Reddy, G.V. and Rodrigues, V. (1999). Sibling cell fate in the Drosophila adult external sense organ lineage is speciﬁed by Prospero function, which is regulated by Numb and Notch. Development 126, 2083–2092. 3551. Reed, B.H. and Orr-Weaver, T.L. (1997). The Drosophila gene morula inhibits mitotic functions in the endo cell cycle and the mitotic cell cycle. Development 124, 3543– 3553. 3552. Reed, C.T., Murphy, C., and Fristrom, D. (1975). The ultrastructure of the differentiating pupal leg of Drosophila melanogaster. W. Roux’s Arch. 178, 285–302. 3553. Reeve, E.C.R. (1960). Some genetic tests on asymmetry of sternopleural chaeta number in Drosophila. Genet. Res. Camb. 1, 151–172. 3554. Reeve, E.C.R. (1961). Modifying the sternopleural hair pattern in Drosophila by selection. Genet. Res. Camb. 2, 158– 160. 3555. Reeve, E.C.R. and Robertson, F.W. (1954). Studies in quantitative inheritance. VI. Sternite chaeta number in Drosophila: A metameric quantitative character. Z. Vererbungslehre 86, 269–288. 3556. Regulski, M., Harding, K., Kostriken, R., Karch, F., Levine, M., and McGinnis, W. (1985). Homeo box genes of the Antennapedia and Bithorax complexes of Drosophila. Cell 43, 71–80.

403

3557. Reh, T.A. and Cagan, R.L. (1994). Intrinsic and extrinsic signals in the developing vertebrate and ﬂy eyes: viewing vertebrate and invertebrate eyes in the same light. Persp. Dev. Neurobiol. 2, 183–190. 3558. Reich, A., Sapir, A., and Shilo, B.-Z. (1999). Sprouty is a general inhibitor of receptor tyrosine kinase signaling. Development 126, 4139–4147. 3559. Reichman-Fried, M., Dickson, B., Hafen, E., and Shilo, B.-Z. (1994). Elucidation of the role of breathless, a Drosophila FGF receptor homolog, in tracheal cell migration. Genes Dev. 8, 428–439. 3560. Reichman-Fried, M. and Shilo, B.-Z. (1995). Breathless, a Drosophila FGF receptor homolog, is required for the onset of tracheal cell migration and tracheole formation. Mechs. Dev. 52, 265–273. 3561. Reichsman, F., Smith, L., and Cumberledge, S. (1996). Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction. J. Cell Biol. 135, 819–827. 3562. Reifegerste, R., Ma, C., and Moses, K. (1997). A polarity ﬁeld is established early in the development of the Drosophila compound eye. Mechs. Dev. 68, 69–79. 3563. Reifegerste, R. and Moses, K. (1999). Genetics of epithelial polarity and pattern in the Drosophila retina. BioEssays 21, 275–285. 3564. Reinhardt, C.A. and Bryant, P.J. (1981). Wound healing in the imaginal discs of Drosophila. II. Transmission electron microscopy of normal and healing wing discs. J. Exp. Zool. 216, 45–61. 3565. Reinhardt, C.A., Hodgkin, N.M., and Bryant, P.J. (1977). Wound healing in the imaginal discs of Drosophila. I. Scanning electron microscopy of normal and healing wing discs. Dev. Biol. 60, 238–257. 3566. Reinhardt, C.A. and Sanchez, L. (1982). Effects of the “sexcombless” chromosome (In(1)sx) on males and females of Drosophila melanogaster. W. Roux’s Arch. 191, 264–269. 3567. Reinitz, J., Kosman, D., Vanario-Alonso, C.E., and Sharp, D.H. (1998). Stripe forming architecture of the gap gene system. Dev. Genet. 23, 11–27. [See also 2001 Nature 411, 151–152.] 3568. Reinke, R., Krantz, D.E., Yen, D., and Zipursky, S.L. (1988). Chaoptin, a cell surface glycoprotein required for Drosophila photoreceptor cell morphogenesis, contains a repeat motif found in yeast and human. Cell 52, 291–301. 3569. Reinke, R. and Zipursky, S.L. (1988). Cell-cell interaction in the Drosophila retina: the bride of sevenless gene is required in photoreceptor cell R8 for R7 cell development. Cell 55, 321–330. 3570. Reiter, C., Schimansky, T., Nie, Z., and Fischbach, K.-F. (1996). Reorganization of membrane contacts prior to apoptosis in the Drosophila retina: the role of the IrreC-rst protein. Development 122, 1931–1940. 3571. Rendel, J.M. (1959). Canalization of the scute phenotype of Drosophila. Evolution 13, 425–439. 3572. Rendel, J.M. (1962). The relationship between gene and phenotype. J. Theor. Biol. 2, 296–308. 3573. Rendel, J.M. (1967). “Canalisation and Gene Control.” Logos Pr./Elek Books, London. 3574. Renfranz, P.J. and Benzer, S. (1989). Monoclonal antibody probes discriminate early and late mutant defects in development of the Drosophila retina. Dev. Biol. 136, 411– 429. 3575. Reppert, S.M. and Sauman, I. (1995). period and timeless tango: a dance of two clock genes. Neuron 15, 983–986.

404

3576. Reuter, R. and Leptin, M. (1994). Interacting functions of snail, twist and huckebein during the early development of germ layers in Drosophila. Development 120, 1137– 1150. 3577. R´evet, B., von Wilcken-Bergmann, B., Bessert, H., Barker, ¨ A., and Muller-Hill, B. (1998). Four dimers of λ repressor bound to suitably spaced pairs of λ operators form octamers and DNA loops over large distances. Curr. Biol. 9, 151–154. 3578. Rhodes, D. and Klug, A. (1993). Zinc ﬁngers. Sci. Am. 268 #2, 56–65. 3579. Rhyu, M.S., Jan, L.Y., and Jan, Y.N. (1994). Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 76, 477–491. 3580. Rhyu, M.S. and Knoblich, J.A. (1995). Spindle orientation and asymmetric cell fate. Cell 82, 523–526. 3581. Ribbeck, K., Kutay, U., Paraskeva, E., and G¨orlich, D. (1998). The translocation of transportin-cargo complexes through nuclear pores is independent of both Ran and energy. Curr. Biol. 9, 47–50. 3582. Richards, A.G. (1951). “The Integument of Arthropods.” Oxford Univ. Pr., London. 3583. Richards, A.G. and Richards, P.A. (1979). The cuticular protuberances of insects. Int. J. Insect Morphol. & Embryol. 8, 143–157. 3584. Richelle, J. and Ghysen, A. (1979). Determination of sensory bristles and pattern formation in Drosophila. I. A model. Dev. Biol. 70, 418–437. 3585. Richter, B., Long, M., Lewontin, R.C., and Nitasaka, E. (1997). Nucleotide variation and conservation at the dpp locus, a gene controlling early development in Drosophila. Genetics 145, 311–323. 3586. Ricos, M.G., Harden, N., Sem, K.P., Lim, L., and Chia, W. (1999). Dcdc42 acts in TGF-β signaling during Drosophila morphogenesis: distinct roles for the Drac1/JNK and Dcdc42/TGF-β cascades in cytoskeletal regulation. J. Cell Sci. 112, 1225–1235. 3587. Riddihough, G. and Ish-Horowicz, D. (1991). Individual stripe regulatory elements in the Drosophila hairy promoter respond to maternal, gap, and pair-rule genes. Genes Dev. 5, 840–854. 3588. Ridley, M. (1999). “Genome: The Autobiography of a Species in 23 Chapters.” Harper-Collins, New York. 3589. Rieckhof, G.E., Casares, F., Ryoo, H.D., Abu-Shaar, M., and Mann, R.S. (1997). Nuclear translocation of Extradenticle requires homothorax, which encodes an Extradenticlerelated homeodomain protein. Cell 91, 171–183. 3590. Riese, D.J., II and Stern, D.F. (1998). Speciﬁcity within the EGF family/ErbB receptor family signaling network. BioEssays 20, 41–48. 3591. Riese, J., Yu, X., Munnerlyn, A., Eresh, S., Hsu, S.-C., Grosschedl, R., and Bienz, M. (1997). LEF-1, a nuclear factor coordinating signaling inputs from wingless and decapentaplegic. Cell 88, 777–787. 3592. Riesgo-Escovar, J.R. and Hafen, E. (1997). Drosophila Jun kinase regulates expression of decapentaplegic via the ETS-domain protein Aop and the AP-1 transcription factor DJun during dorsal closure. Genes Dev. 11, 1717–1727. 3593. Riesgo-Escovar, J.R., Jenni, M., Fritz, A., and Hafen, E. (1996). The Drosophila Jun-N-terminal kinase is required for cell morphogenesis but not for DJun-dependent cell fate speciﬁcation in the eye. Genes Dev. 10, 2759– 2768. 3594. Rietdorf, J., Siegert, F., Dharmawardhane, S., Firtel, R.A.,

REFERENCES

3595.

3596.

3597.

3598.

3599.

3600.

3601.

3602.

3603.

3604.

3605.

3606.

3607.

3608.

3609.

3610. 3611.

and Weijer, C.J. (1997). Analysis of cell movement and signalling during ring formation in an activated Gα1 mutant of Dictyostelium discoideum that is defective in prestalk zone formation. Dev. Biol. 181, 79–90. Rietveld, A., Neutz, S., Simons, K., and Eaton, S. (1999). Association of sterol- and glycosylphosphatidylinositollinked proteins with Drosophila raft lipid microdomains. J. Biol. Chem. 274 #17, 12049–12054. Riezman, H., Woodman, P.G., van Meer, G., and Marsh, M. (1997). Molecular mechanisms of endocytosis. Cell 91, 731–738. Riggleman, B., Schedl, P., and Wieschaus, E. (1990). Spatial expression of the Drosophila segment polarity gene armadillo is posttranscriptionally regulated by wingless. Cell 63, 549–560. Riggleman, B., Wieschaus, E., and Schedl, P. (1989). Molecular analysis of the armadillo locus: uniformly distributed transcripts and a protein with novel internal repeats are associated with a Drosophila segment polarity gene. Genes Dev. 3, 96–113. Rijsewijk, F., Schuermann, M., Wagenaar, E., Parren, P., Weigel, D., and Nusse, R. (1987). The Drosophila homolog of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless. Cell 50, 649–657. Rinc´on-Limas, D.E., Lu, C.-H., Canal, I., and Botas, J. (2000). The level of DLDB/CHIP controls the activity of the LIM homeodomain protein Apterous: evidence for a functional tetramer complex in vivo. EMBO J. 19, 2602– 2614. Rinc´on-Limas, D.E., Lu, C.-H., Canal, I., Calleja, M., ´ Rodr´ıguez-Esteban, C., Izpisua-Belmonte, J.C., and Botas, J. (1999). Conservation of the expression and function of apterous orthologs in Drosophila and mammals. Proc. Natl. Acad. Sci. USA 96, 2165–2170. Ripoll, P. (1972). The embryonic organization of the imaginal wing disc of Drosophila melanogaster. W. Roux’ Arch. 169, 200–215. Ripoll, P., El Messal, M., Laran, E., and Simpson, P. (1988). A gradient of afﬁnities for sensory bristles across the wing blade of Drosophila melanogaster. Development 103, 757– 767. Ritossa, F. (1976). The bobbed locus. In “The Genetics and Biology of Drosophila,” Vol. 1b (M. Ashburner and E. Novitski, eds.). Acad. Pr.: New York, pp. 801–846. Rivera-Pomar, R. and J¨ackle, H. (1996). From gradients to stripes in Drosophila embryogenesis: ﬁlling in the gaps. Trends Genet. 12, 478–483. Rivera-Pomar, R., Niessing, D., Schmidt-Ott, U., Gehring, W.J., and J¨ackle, H. (1996). RNA binding and translational suppression by bicoid. Nature 379, 746–749. Rives, A.F., Wehrli, M., and DiNardo, S. (2001). The role of arrow in the wingless signaling pathway. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a207. Rivlin, P.K., Gong, A., Schneiderman, A.M., and Booker, R. (2001). The role of Ultrabithorax in the patterning of adult thoracic muscles in Drosophila melanogaster. Dev. Genes Evol. 211, 55–66. Rivlin, P.K., Schneiderman, A.M., and Booker, R. (2000). Imaginal pioneers preﬁgure the formation of adult thoracic muscles in Drosophila melanogaster. Dev. Biol. 222, 450–459. Rivlin, R. (1986). “The Algorithmic Image: Graphic Visions of the Computer Age.” Microsoft Pr., Richmond, Wash. Roark, M., Sturtevant, M.A., Emery, J., Vaessin, H., Grell, E., and Bier, E. (1995). scratch, a pan-neural gene encoding

REFERENCES

3612.

3613.

3614.

3615.

3616.

3617.

3618.

3619. 3620.

3621.

3622. 3623.

3624.

3625.

3626.

3627.

3628.

3629.

3630.

a zinc-ﬁnger protein related to snail, promotes neuronal development. Genes Dev. 9, 2384–2398. Robbins, D.J., Nybakken, K.E., Kobayashi, R., Sisson, J.C., Bishop, J.M., and Th´erond, P.P. (1997). Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein Costal2. Cell 90, 225–234. Roberts, P. (1961). Bristle formation controlled by the achaete locus in genetic mosaics of Drosophila melanogaster. Genetics 46, 1241–1243. Roberts, P. (1964). Mosaics involving aristapedia, a homeotic mutant of Drosophila melanogaster. Genetics 49, 593–598. Roberts, P.A. (1976). The genetics of chromosome aberration. In “The Genetics and Biology of Drosophila,” Vol. 1a (M. Ashburner and E. Novitski, eds.). Acad. Pr.: New York, pp. 67–184. Roberts, P.A. and Broderick, D.J. (1982). Properties and evolutionary potential of newly induced tandem duplications in Drosophila melanogaster. Genetics 102, 75– 89. Robertson, F.W. (1959). Studies in quantitative inheritance. XII. Cell size and number in relation to genetic and environmental variation of body size in Drosophila. Genetics 44, 869–896. Robertson, H.M. (2001). Molecular evolution of the insect chemoreceptor superfamily and underannotation of the Drosophila genome. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a327. Robertson, M. (1981). Gene families, hopeful monsters and the selﬁsh genetics of DNA. Nature 293, 333–334. Robinow, S., Campos, A.R., Yao, K.-M., and White, K. (1988). The elav gene product of Drosophila, required in neurons, has three RNP consensus motifs. Science 242, 1570–1572. Robinow, S., Draizen, T.A., and Truman, J.W. (1997). Genes that induce apoptosis: transcriptional regulation in identiﬁed, doomed neurons of the Drosophila CNS. Dev. Biol. 190, 206–213. Robinson, M.J. and Cobb, M.H. (1997). Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol. 9, 180–186. Roch, F. and Akam, M. (2000). Ultrabithorax and the control of cell morphology in Drosophila halteres. Development 127, 97–107. Roch, F., Baonza, A., Mart´ın-Blanco, E., and Garc´ıa-Bellido, A. (1998). Genetic interactions and cell behaviour in blistered mutants during proliferation and differentiation of the Drosophila wing. Development 125, 1823–1832. Rodgers, M.E. and Shearn, A. (1977). Patterns of protein synthesis in imaginal discs of Drosophila melanogaster. Cell 12, 915–921. Rodrigues, V. (1988). Spatial coding of olfactory information in the antennal lobe of Drosophila melanogaster. Brain Res. 453, 299–307. Rodriguez, I. and Basler, K. (1997). Control of compartmental afﬁnity boundaries by Hedgehog. Nature 389, 614– 618. Rodr´ıguez, I., Hern´andez, R., Modolell, J., and RuizG´omez, M. (1990). Competence to develop sensory organs is temporally and spatially regulated in Drosophila epidermal primordia. EMBO J. 9, 3583–3592. Roegiers, F., Younger-Shepherd, S., Jan, L.Y., and Jan, Y.N. (2001). Genetic analysis of two types of asymmetric divisions of sensory organ precursor cell lineage. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a50. Roegiers, F., Younger-Shepherd, S., Jan, L.Y., and Jan,

405

3631.

3632.

3633.

3634.

3635.

3636.

3637.

3638. 3639.

3640.

3641.

3642.

3643.

3644.

3645. 3646.

3647.

3648.

Y.N. (2001). Two types of asymmetric divisions in the Drosophila sensory organ precursor cell lineage. Nature Cell Biol. 3, 58–67. [Cf. 2001 Dev. Biol. 240, 361–376.] Rogers, B.T. and Kaufman, T.C. (1997). Structure of the insect head in ontogeny and phylogeny: a view from Drosophila. Int. Rev. Cytol. 174, 1–84. Rogers, S., Wells, R., and Rechsteiner, M. (1986). Amino acid sequences common to rapidly degraded proteins: the PEST Hypothesis. Science 234, 364–368. Rogge, R., Cagan, R., Majumdar, A., Dulaney, T., and Banerjee, U. (1992). Neuronal development in the Drosophila retina: the sextra gene deﬁnes an inhibitory component in the developmental pathway of R7 photoreceptor cells. Proc. Natl. Acad. Sci. USA 89, 5271–5275. Rogge, R., Green, P.J., Urano, J., Horn-Saban, S., Mlodzik, M., Shilo, B.-Z., Hartenstein, V., and Banerjee, U. (1995). The role of yan in mediating the choice between cell division and differentiation. Development 121, 3947–3958. Rogge, R.D., Karlovich, C.A., and Banerjee, U. (1991). Genetic dissection of a neurodevelopmental pathway: Son of sevenless functions downstream of the sevenless and EGF receptor tyrosine kinases. Cell 64, 39–48. Roignant, J.Y., Faucheux, M., Lepesant, J.-A., Th´eodore, L., and Antoniewski, C. (2001). Identiﬁcation of Batman as a putative partner of the Broad proteins. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a97. Romani, S., Campuzano, S., Macagno, E.R., and Modolell, J. (1989). Expression of achaete and scute genes in Drosophila imaginal discs and their function in sensory organ development. Genes Dev. 3, 997–1007. Rommel, C. and Hafen, E. (1998). Ras–a versatile cellular switch. Curr. Opin. Gen. Dev. 8, 412–418. Rommel, C., Radziwill, G., Moelling, K., and Hafen, E. (1997). Negative regulation of Raf activity by binding of 14-3-3 to the amino terminus of Raf in vivo. Mechs. Dev. 64, 95–104. Rooke, J., Pan, D., Xu, T., and Rubin, G.M. (1996). KUZ, a conserved metalloprotease-disintegrin protein with two roles in Drosophila neurogenesis. Science 273, 1227– 1231. Rooke, J.E. and Xu, T. (1998). Positive and negative signals between interacting cells for establishing neural fate. BioEssays 20, 209–214. Rørth, P. and Montell, D.J. (1992). Drosophila C/EBP: a tissue-speciﬁc DNA-binding protein required for embryonic development. Genes Dev. 6, 2299–2311. Rørth, P., Szabo, K., Bailey, A., Laverty, T., Rehm, J., Rubin, G.M., Weigmann, K., Mil´an, M., Benes, V., Ansorge, W., and Cohen, S.M. (1998). Systematic gain-of-function genetics in Drosophila. Development 125, 1049–1057. Rose, S.M. (1952). A hierarchy of self-limiting reactions as the basis of cellular differentiation and growth control. Am. Nat. 86, 337–354. Rose, S.M. (1957). Cellular interaction during differentiation. Biol. Rev. 32, 351–382. Rose, S.M. (1970). Differentiation during regeneration caused by migration of repressors in bioelectric ﬁelds. Amer. Zool. 10, 91–99. Roseland, C.R. and Schneiderman, H.A. (1979). Regulation and metamorphosis of the abdominal histoblasts of Drosophila melanogaster. W. Roux’s Arch. 186, 235–265. Rosenberg, M.I., Phippen, T.M., Secombe, J., Sweigart, A.L., and Parkhurst, S.M. (2001). Cofactors for Hairymediated repression. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a125.

406

3649. Rosenfeld, M.G. (1991). POU-domain transcription factors: pou-er-ful developmental regulators. Genes Dev. 5, 897–907. 3650. Rosin-Arbesfeld, R., Townsley, F., and Bienz, M. (2000). The APC tumor suppressor has a nuclear export function. Nature 406, 1009–1012. 3651. Rossky, P.J. (2001). Molecules at the edge. Nature 410, 645– 648. 3652. Rothe, M., Pehl, M., Taubert, H., and J¨ackle, H. (1992). Loss of gene function through rapid mitotic cycles in the Drosophila embryo. Nature 359, 156–159. 3653. Rothe, M., Wimmer, E.A., Pankratz, M.J., Gonz´alez-Gait´an, M., and J¨ackle, H. (1994). Identical transacting factor requirement for knirps and knirps-related gene expression in the anterior but not in the posterior region of the Drosophila embryo. Mechs. Dev. 46, 169–181. 3654. Rothenﬂuh, A., Young, M.W., and Saez, L. (2000). A TIMELESS-independent function for PERIOD proteins in the Drosophila clock. Neuron 26, 505–514. 3655. Roush, W. (1995). Sifting mitosis, cell fate in ﬂy eyes. Science 270, 916–917. 3656. Roush, W. (1996). Receptor for vital protein ﬁnally found. Science 273, 309. 3657. Roush, W. (1996). “Smart” genes use many cues to set cell fate. Science 272, 652–653. 3658. Roush, W. (1997). A “master control” gene for ﬂy eyes shares its power. Science 275, 618–619. 3659. Rousset, R., Mack, J.A., Wharton, K.A., Jr., Axelrod, J.D., Cadigan, K.M., Fish, M.P., Nusse, R., and Scott, M.P. (2001). naked cuticle targets dishevelled to antagonize Wnt signal transduction. Genes Dev. 15, 658–671. 3660. Roy, S. and VijayRaghavan, K. (1997). Homeotic genes and the regulation of myoblast migration, fusion, and ﬁbre-speciﬁc gene expression during adult myogenesis in Drosophila. Development 124, 3333–3341. 3661. Roy, S. and VijayRaghavan, K. (1999). Muscle pattern diversiﬁcation in Drosophila: the story of imaginal myogenesis. BioEssays 21, 486–498. 3662. Royet, J., Bouwmeester, T., and Cohen, S.M. (1998). Notchless encodes a novel WD40-repeat-containing protein that modulates Notch signaling activity. EMBO J. 17, 7351– 7360. 3663. Royet, J. and Finkelstein, R. (1995). Pattern formation in Drosophila head development: the role of the orthodenticle homeobox gene. Development 121, 3561–3572. 3664. Royet, J. and Finkelstein, R. (1996). hedgehog, wingless and orthodenticle specify adult head development in Drosophila. Development 122, 1849–1858. 3665. Royet, J. and Finkelstein, R. (1997). Establishing primordia in the Drosophila eye-antennal imaginal disc: the roles of decapentaplegic, wingless, and hedgehog. Development 124, 4793–4800. 3666. Rozovskaia, T., Rozenblatt-Rosen, O., Sedkov, Y., Burakov, D., Yano, T., Nakamura, T., Petruk, S., Ben-Simchon, L., Croce, C.M., Mazo, A., and Canaani, E. (2000). Selfassociation of the SET domains of human ALL-1 and of Drosophila TRITHORAX and ASH1 proteins. Oncogene 19, 351–357. 3667. Rozovskaia, T., Tillib, S., Smith, S., Sedkov, Y., RozenblattRosen, O., Petruk, S., Yano, T., Nakamura, T., Ben-Simchon, L., Gildea, J., Croce, C.M., Shearn, A., Canaani, E., and Mazo, A. (1999). Trithorax and ASH1 interact directly and associate with the trithorax group-responsive bxd region of the Ultrabithorax promoter. Mol. Cell. Biol. 19, 6441– 6447.

REFERENCES

3668. Ruberte, E., Marty, T., Nellen, D., Affolter, M., and Basler, K. (1995). An absolute requirement for both the type II and type I receptors, punt and thick veins, for Dpp signaling in vivo. Cell 80, 889–897. 3669. Rubin, G.M. (1988). Drosophila melanogaster as an experimental organism. Science 240, 1453–1459. 3670. Rubin, G.M. (1989). Development of the Drosophila retina: inductive events studied at single cell resolution. Cell 57, 519–520. 3671. Rubin, G.M. (1991). Signal transduction and the fate of the R7 photoreceptor in Drosophila. Trends Genet. 7, 372–377. 3672. Rubin, G.M., Chang, H.C., Karim, F., Laverty, T., Michaud, N.R., Morrison, D.K., Rebay, I., Tang, A., Therrien, M., and Wassarman, D.A. (1997). Signal transduction downstream from RAS in Drosophila. Cold Spr. Harb. Symp. Quant. Biol. 62, 347–352. 3673. Rubin, G.M. and Lewis, E.B. (2000). A brief history of Drosophila’s contributions to genome research. Science 287, 2216–2218. 3674. Rubin, G.M., Yandell, M.D., Wortman, J.R., Gabor Miklos, G.L., Nelson, C.R., Hariharan, I.K., Fortini, M.E., Li, P.W., Apweiler, R., Fleischmann, W., Cherry, J.M., Henikoff, S., Skupski, M.P., Misra, S., Ashburner, M., Birney, E., Boguski, M.S., Brody, T., Brokstein, P., Celniker, S.E., Chervitz, S.A., Coates, D., Cravchik, A., Gabrielian, A., Galle, R.F., Gelbart, W.M., George, R.A., Goldstein, L.S.B., Gong, F., Guan, P., Harris, N.L., Hay, B.A., Hoskins, R.A., Li, J., Li, Z., Hynes, R.O., Jones, S.J.M., Kuehl, P.M., Lemaitre, B., Littleton, J.T., Morrison, D.K., Mungall, C., O’Farrell, P.H., Pickeral, O.K., Shue, C., Vosshall, L.B., Zhang, J., Zhao, Q., Zheng, X.H., Zhong, F., Zhong, W., Gibbs, R., Venter, J.C., Adams, M.D., and Lewis, S. (2000). Comparative genomics of the eukaryotes. Science 287, 2204–2215. 3675. Rubinfeld, B., Albert, I., Porﬁri, E., Fiol, C., Munemitsu, S., and Polakis, P. (1996). Binding of GSK3β to the APCβ-catenin complex and regulation of complex assembly. Science 272, 1023–1026. 3676. Rudd, C.E. (1999). Adaptors and molecular scaffolds in immune cell signaling. Cell 96, 5–8. 3677. Ruddle, F.H., Bartels, J.L., Bentley, K.L., Kappen, C., Murtha, M.T., and Pendleton, J.W. (1994). Evolution of Hox genes. Annu. Rev. Genet. 28, 423–442. 3678. Ruden, D.M., Wang, X., Cui, W., Mori, D., and Alterman, M. (1999). A novel follicle-cell-dependent dominant female sterile allele, StarKojak, alters receptor tyrosine kinase signaling in Drosophila. Dev. Biol. 207, 393–407. ¨ 3679. Rudiger, M. (1998). Vinculin and α-catenin: shared and unique functions in adherens junctions. BioEssays 20, 733–740. 3680. Rudolph, K.M., Liaw, G.-J., Daniel, A., Green, P., Courey, A.J., Hartenstein, V., and Lengyel, J.A. (1997). Complex regulatory region mediating tailless expression in early embryonic patterning and brain development. Development 124, 4297–4308. 3681. Ruel, L., Bourouis, M., Heitzler, P., Pantesco, V., and Simpson, P. (1993). Drosophila shaggy kinase and rat glycogen synthase kinase-3 have conserved activities and act downstream of Notch. Nature 362, 557–560. 3682. Ruel, L., Pantesco, V., Lutz, Y., Simpson, P., and Bourouis, M. (1993). Functional signiﬁcance of a family of protein kinases encoded at the shaggy locus in Drosophila. EMBO J. 12, 1657–1669. 3683. Ruel, L., Stambolic, V., Ali, A., Manoukian, A.S., and Woodgett, J.R. (1999). Regulation of the protein kinase activity of ShaggyZeste-white3 by components of the Wingless

REFERENCES

3684.

3685. 3686. 3687. 3688.

3689.

3690.

3691.

3692.

3693.

3694.

3695.

3696.

3697.

3698.

3699.

3700.

3701.

3702.

3703.

pathway in Drosophila cells and embryos. J. Biol. Chem. 274 #31, 21790–21796. [See also 2001 Cell 105, 721–732.] Ruiz G´omez, M. and Bate, M. (1997). Segregation of myogenic lineages in Drosophila requires Numb. Development 124, 4857–4866. Ruiz i Altaba, A. (1997). Catching a Gli-mpse of Hedgehog. Cell 90, 193–196. Ruiz i Altaba, A. (1999). Gli proteins and Hedgehog signaling. Trends Genet. 15, 418–425. Ruiz-G´omez, M. (1998). Muscle patterning and speciﬁcation in Drosophila. Int. J. Dev. Biol. 42, 283–290. Ruiz-G´omez, M. and Modolell, J. (1987). Deletion analysis of the achaete-scute locus of Drosophila melanogaster. Genes Dev. 1, 1238–1246. Rulifson, E.J. and Blair, S.S. (1995). Notch regulates wingless expression and is not required for reception of the paracrine wingless signal during wing margin neurogenesis in Drosophila. Development 121, 2813–2824. Rulifson, E.J., Micchelli, C.A., Axelrod, J.D., Perrimon, N., and Blair, S.S. (1996). wingless reﬁnes its own expression domain on the Drosophila wing margin. Nature 384, 72–74 (erratum: 1996 Nature 384: 597). Rulifson, E.J., Wu, C.-H., and Nusse, R. (2000). Pathway speciﬁcity by the bifunctional receptor Frizzled is determined by afﬁnity for Wingless. Molec. Cell 6, 117–126. Ruohola-Baker, H., Grell, E., Chou, T.-B., Baker, D., Jan, L.Y., and Jan, Y.N. (1993). Spatially localized Rhomboid is required for establishment of the dorsal-ventral axis in Drosophila oogenesis. Cell 73, 953–965. Rusch, J. and Levine, M. (1997). Regulation of a dpp target gene in the Drosophila embryo. Development 124, 303– 311. Rusconi, J.C. and Corbin, V. (1998). Evidence for a novel Notch pathway required for muscle precursor selection in Drosophila. Mechs. Dev. 79, 39–50. Rusconi, J.C. and Corbin, V. (1999). A widespread and early requirement for a novel Notch function during Drosophila embryogenesis. Dev. Biol. 215, 388–398. Rushlow, C., Colosimo, P.F., Lin, M.-c., Xu, M., and Kirov, N. (2001). Transcriptional regulation of the Drosophila gene zen by competing Smad and Brinker inputs. Genes Dev. 15, 340–351. Rushlow, C.A., Hogan, A., Pinchin, S.M., Howe, K.M., Lardelli, M., and Ish-Horowicz, D. (1989). The Drosophila hairy protein acts in both segmentation and bristle patterning and shows homology to N-myc. EMBO J. 8, 3095– 3103. Rushton, E., Drysdale, R., Abmayr, S.M., Michelson, A.M., and Bate, M. (1995). Mutations in a novel gene, myoblast city, provide evidence in support of the founder cell hypothesis for Drosophila muscle development. Development 121, 1979–1988. Russell, E.S. (1917). “Form and Function: A Contribution to the History of Animal Morphology.” E. P. Dutton, New York. Russell, J., Gennissen, A., and Nusse, R. (1992). Isolation and expression of two novel Wnt/wingless gene homologues in Drosophila. Development 115, 475–485. Russell, M. (1982). Imaginal discs. In “A Handbook of Drosophila Development,” (R. Ransom, ed.). Elsevier: Amsterdam, pp. 95–121. Russell, M.A. (1974). Pattern formation in the imaginal discs of a temperature-sensitive cell-lethal mutant of Drosophila melanogaster. Dev. Biol. 40, 24–39. Russell, M.A. (1985). Positional information in imaginal

407

3704. 3705.

3706.

3707.

3708.

3709.

3710.

3711.

3712.

3713.

3714.

3715.

3716.

3717.

3718.

3719.

discs: A Cartesian coordinate model. In “Mathematical Essays on Growth and the Emergence of Form,” (P.L. Antonelli, ed.). Univ. of Alberta Pr.: Edmonton, pp. 169–183. Russell, M.A. (1985). Positional information in insect segments. Dev. Biol. 108, 269–283. Russell, M.A., Girton, J.R., and Morgan, K. (1977). Pattern formation in a ts-cell-lethal mutant of Drosophila: the range of phenotypes induced by larval heat treatments. W. Roux’s Arch. 183, 41–59. Russell, S. and Ashburner, M. (1996). Ecdysone-regulated chromosome pufﬁng in Drosophila melanogaster. In “Metamorphosis: Postembryonic Reprogramming of Gene Expression in Amphibian and Insect Cells,” (L.I. Gilbert, J.R. Tata, and B.G. Atkinson, ed.). Acad. Pr.: New York, pp. 109–144. Russell, S.R.H., Sanchez-Soriano, N., Wright, C.R., and Ashburner, M. (1996). The Dichaete gene of Drosophila melanogaster encodes a SOX-domain protein required for embryonic segmentation. Development 122, 3669–3676. Rusten, T.E., Cantera, R., Urban, J., Technau, G., Kafatos, F.C., and Barrio, R. (2001). Spalt modiﬁes EGFR-mediated induction of chordotonal precursors in the embryonic PNS of Drosophila promoting the development of oenocytes. Development 128, 711–722. Rutherford, S.L. (2000). From genotype to phenotype: buffering mechanisms and the storage of genetic information. BioEssays 22, 1095–1105. Rutila, J.E., Suri, V., Le, M., So, W.V., Rosbash, M., and Hall, J.C. (1998). CYCLE is a second bHLH-PAS clock protein essential for circadian rhythmicity and transcription of Drosophila period and timeless. Cell 93, 805–814. Rutledge, B.J., Zhang, K., Bier, E., Jan, Y.N., and Perrimon, N. (1992). The Drosophila spitz gene encodes a putative EGF-like growth factor involved in dorsal-ventral axis formation and neurogenesis. Genes Dev. 6, 1503–1517. Ruvkin, G. and Finney, M. (1991). Regulation of transcription and cell identity by POU domain proteins. Cell 64, 475–478. Ryan, A.K. and Rosenfeld, M.G. (1997). POU domain family values: ﬂexibility, partnerships, and developmental codes. Genes Dev. 11, 1207–1225. Rybtsova, N., Waltzer, L., and Haenlin, M. (2001). Searching for new proneural genes which might regulate the activity of the pan-neural genes deadpan, scratch and snail in Drosophila. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a267. Ryoo, H.D. and Mann, R.S. (1999). The control of trunk Hox speciﬁcity and activity by Extradenticle. Genes Dev. 13, 1704–1716. Ryoo, H.D., Marty, T., Casares, F., Affolter, M., and Mann, R.S. (1999). Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex. Development 126, 5137–5148. [See also 2001 Development 128, 3405–3413.] Sackerson, C., Fujioka, M., and Goto, T. (1999). The evenskipped locus is contained in a 16-kb chromatin domain. Dev. Biol. 211, 39–52. Saebøe-Larssen, S., Lyamouri, M., Merriam, J., Oksvold, M.P., and Lambertsson, A. (1998). Ribosomal protein insufﬁciency and the Minute syndrome in Drosophila: a dose-response relationship. Genetics 148, 1215–1224. Saenz-Robles, M.T., Maschat, F., Tabata, T., Scott, M.P., and Kornberg, T.B. (1995). Selection and characterization of sequences with high afﬁnity for the engrailed protein of Drosophila. Mechs. Dev. 53, 185–195.

408

3720. Saez, L. and Young, M.W. (1996). Regulation of nuclear entry of the Drosophila clock proteins Period and Timeless. Neuron 17, 911–920. 3721. Saget, O., Forquignon, F., Santamaria, P., and Randsholt, N.B. (1998). Needs and targets for the multi sex combs gene product in Drosophila melanogaster. Genetics 149, 1823– 1838. 3722. Sahut-Barnola, I., Godt, D., Laski, F.A., and Couderc, J.-L. (1995). Drosophila ovary morphogenesis: analysis of terminal ﬁlament formation and identiﬁcation of a gene required for this process. Dev. Biol. 170, 127– 135. 3723. Saint, R., Kalionis, B., Lockett, T.J., and Elizur, A. (1988). Pattern formation in the developing eye of Drosophila melanogaster is regulated by the homeo-box gene, rough. Nature 334, 151–154. 3724. Sakanaka, C., Leong, P., Xu, L., Harrison, S.D., and Williams, L.T. (1999). Casein kinase 1 in the Wnt pathway: regulation of β-catenin function. Proc. Natl. Acad. Sci. USA 96, 12548–12552. 3725. Salcini, A.E., Confalonieri, S., Doria, M., Santolini, E., Tassi, E., Minenkova, O., Cesareni, G., Pelicci, P.G., and Di Fiore, P.P. (1997). Binding speciﬁcity and in vivo targets of the EH domain, a novel protein-protein interaction module. Genes Dev. 11, 2239–2249. 3726. Salecker, I., Clandinin, T.R., and Zipursky, S.L. (1998). Hedgehog and Spitz: making a match between photoreceptor axons and their targets. Cell 95, 587–590. 3727. Salser, S.J. and Kenyon, C. (1996). A C. elegans Hox gene switches on, off, on and off again to regulate proliferation, differentiation and morphogenesis. Development 122, 1651–1661. 3728. Salzberg, A., D’Evelyn, D., Schulze, K.L., Lee, J.-K., Strumpf, D., Tsai, L., and Bellen, H.J. (1994). Mutations affecting the pattern of the PNS in Drosophila reveal novel aspects of neuronal development. Neuron 13, 269–287. 3729. Samakovlis, C., Hacohen, N., Manning, G., Sutherland, D.C., Guillemin, K., and Krasnow, M.A. (1996). Development of the Drosophila tracheal system occurs by a series of morphologically distinct but genetically coupled branching events. Development 122, 1395– 1407. 3730. Sampedro, J., Johnston, P., and Lawrence, P.A. (1993). A role for wingless in the segmental gradient of Drosophila? Development 117, 677–687. 3731. Samson, M.-L. (1998). Evidence for 3 untranslated regiondependent autoregulation of the Drosophila gene encoding the neuronal nuclear RNA-binding protein ELAV. Genetics 150, 723–733. 3732. S´anchez, L., Casares, F., Gorﬁnkiel, N., and Guerrero, I. (1997). The genital disc of Drosophila melanogaster. II. Role of the genes hedgehog, decapentaplegic and wingless. Dev. Genes Evol. 207, 229–241. [See also 2001 BioEssays 23, 698–707.] 3733. S´anchez, L., Gorﬁnkiel, N., and Guerrero, I. (2001). Sex determination genes control the development of the Drosophila genital disc, modulating the response to Hedgehog, Wingless and Decapentaplegic signals. Development 128, 1033–1043. 3734. S´anchez, L. and N¨othiger, R. (1983). Sex determination and dosage compensation in Drosophila melanogaster: production of male clones in XX females. EMBO J. 2, 485– 491. 3735. S´anchez-Garc´ıa, I., Osada, H., Forster, A., and Rabbitts, T.H. (1993). The cysteine-rich LIM domains inhibit DNA

REFERENCES

3736.

3737. 3738.

3739.

3740.

3741.

3742.

3743.

3744.

3745.

3746.

3747.

3748.

3749.

3750. 3751.

3752.

3753.

3754.

3755.

binding by the associated homeodomain in Isl-1. EMBO J. 12, 4243–4250. S´anchez-Garc´ıa, I. and Rabbitts, T.H. (1994). The LIM domain: a new structural motif found in zinc-ﬁnger-like proteins. Trends Genet. 10, 315–320. S´anchez-Herrero, E. (1988). Heads or tails?: a homeotic gene for both. Trends Genet. 4, 119–120. S´anchez-Herrero, E. (1991). Control of the expression of the bithorax complex genes abdominal-A and AbdominalB by cis-regulatory regions in Drosophila embryos. Development 111, 437–449. S´anchez-Herrero, E., Couso, J.P., Capdevila, J., and Guerrero, I. (1996). The fu gene discriminates between pathways to control dpp expression in Drosophila imaginal discs. Mechs. Dev. 55, 159–170. S´anchez-Herrero, E., Vern´os, I., Marco, R., and Morata, G. (1985). Genetic organization of Drosophila bithorax complex. Nature 313, 108–113. Sander, K. (1991). Wilhelm Roux and the rest: developmental theories 1885–1895. Roux’s Arch. Dev. Biol. 200, 297–299. Sander, K. (1992). Hans Driesch the critical mechanist: “Analytische Theorie der organischen Entwicklung”. Roux’s Arch. Dev. Biol. 201, 331–333. Sander, K. (1993). Entelechy and the ontogenetic machine–work and views of Hans Driesch from 1895 to 1910. Roux’s Arch. Dev. Biol. 202, 67–69. Sander, K. (1994). Of gradients and genes: developmental concepts of Theodor Boveri and his students. Roux’s Arch. Dev. Biol. 203, 295–297. Sander, K. (1996). Hilde Mangold (1898–1924) and Spemann’s organizer: achievement and tragedy. Roux’s Arch. Dev. Biol. 205, 323–332. Sang, J.H. (1961). Environmental control of mutant expression. In “Insect Polymorphism,” Symp. Roy. Entomol. Soc. Lond., Vol. 1 (J.S. Kennedy, ed.). E. W. Classey: Farington, Oxon, U.K., pp. 91–102. Sanicola, M., Sekelsky, J., Elson, S., and Gelbart, W.M. (1995). Drawing a stripe in Drosophila imaginal disks: negative regulation of decapentaplegic and patched expression by engrailed. Genetics 139, 745–756. Sanson, B., Alexandre, C., Fascetti, N., and Vincent, J.-P. (1999). Engrailed and Hedgehog make the range of Wingless asymmetric in Drosophila embryos. Cell 98, 207– 216. Sanson, B., White, P., and Vincent, J.-P. (1996). Uncoupling cadherin-based adhesion from wingless signalling in Drosophila. Nature 383, 627–630. Santamaria, P. (1983). Analysis of haploid mosaics in Drosophila. Dev. Biol. 96, 285–295. Santamar´ıa, P. (1979). Heat shock induced phenocopies of dominant mutants of the Bithorax Complex in Drosophila melanogaster. Mol. Gen. Genet. 172, 161–163. Santamar´ıa, P. (1998). Genesis versus epigenesis: the odd jobs of the Polycomb group of genes. Int. J. Dev. Biol. 42, 463–469. Santamaria, P., Deatrick, J., and Randsholt, N.B. (1989). Pattern triplications following genetic ablation on the wing of Drosophila: Effect of eliminating the polyhomeotic gene. Roux’s Arch. Dev. Biol. 198, 65–77. Santamar´ıa, P. and Gans, M. (1980). Chimaeras of Drosophila melanogaster obtained by injection of haploid nuclei. Nature 287, 143–144. Santamar´ıa, P. and Randsholt, N.B. (1995). Characterization of a region of the X chromosome of Drosophila

REFERENCES

3756.

3757.

3758.

3759.

3760.

3761.

3762.

3763.

3764.

3765. 3766.

3767. 3768.

3769. 3770.

3771.

3772.

3773.

including multi sex combs (mxc), a Polycomb group gene which also functions as a tumour suppressor. Mol. Gen. Genet. 246, 282–290. Santar´en, J.F., Assiego, R., and Garc´ıa-Bellido, A. (1993). Patterns of protein synthesis in the imaginal discs of Drosophila melanogaster : a comparison between different discs and stages. Roux’s Arch. Dev. Biol. 203, 131– 139. Santolini, E., Puri, C., Salcini, A.E., Gagliani, M.C., Pelicci, P.G., Tacchetti, C., and Di Fiore, P.P. (2000). Numb is an endocytic protein. J. Cell Biol. 151, 1345–1351. Sapir, A., Schweitzer, R., and Shilo, B.-Z. (1998). Sequential activation of the EGF receptor pathway during Drosophila oogenesis establishes the dorsoventral axis. Development 125, 191–200. Sasai, Y., Kageyama, R., Tagawa, Y., Shigemoto, R., and Nakanishi, S. (1992). Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split. Genes Dev. 6, 2620–2634. Sass, G., Fehr, K., and Lucchesi, J.C. (2001). The chromatinremodeling MSL complex of Drosophila spreads in cis to activated loci on the X chromosome. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a89. Sathe, S.S. and Harte, P.J. (1995). The Drosophila extra sex combs protein contains WD motifs essential for its function as a repressor of homeotic genes. Mechs. Dev. 52, 77– 87. Sato, A., Kojima, T., Ui-Tei, K., Miyata, Y., and Saigo, K. (1999). Dfrizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic mutants. Development 126, 4421–4430. Sato, M., Kojima, T., Michiue, T., and Saigo, K. (1999). Bar homeobox genes are latitudinal prepattern genes in the developing Drosophila notum whose expression is regulated by the concerted functions of decapentaplegic and wingless. Development 126, 1457–1466. Sato, M. and Saigo, K. (2000). Involvement of pannier and u-shaped in regulation of Decapentaplegic-dependent wingless expression in developing Drosophila notum. Mechs. Dev. 93, 127–138. Sato, T. (1984). A new homoeotic mutation affecting antennae and legs. Dros. Info. Serv. 60, 180–182. Sato, T. and Denell, R.E. (1985). Homoeosis in Drosophila: anterior and posterior transformations of Polycomb lethal embryos. Dev. Biol. 110, 53–64. Satoh, N. (1982). Timing mechanisms in early embryonic development. Differentiation 22, 156–163. Sauer, F. and J¨ackle, H. (1995). Heterodimeric Drosophila gap gene protein complexes acting as transcriptional repressors. EMBO J. 14, 4773–4780. Sauer, R.T. (1990). Scissors and helical forks. Nature 347, 514–515. Saulier-Le Dr´ean, B., Nasiadka, A., Dong, J., and Krause, H.M. (1998). Dynamic changes in the functions of Oddskipped during early Drosophila embryogenesis. Development 125, 4851–4861. Sawamoto, K., Okabe, M., Tanimura, T., Mikoshiba, K., Nishida, Y., and Okano, H. (1996). The Drosophila secreted protein Argos regulates signal transduction in the Ras/MAPK pathway. Dev. Biol. 178, 13–22. Sawamoto, K., Okano, H., Kobayakawa, Y., Hayashi, S., Mikoshiba, K., and Tanimura, T. (1994). The function of argos in regulating cell fate decisions during Drosophila eye and wing vein development. Dev. Biol. 164, 267–276. Scandalios, J.G. and Wright, T.R.F., eds. (1990). “Genetic

409

3774.

3775.

3776.

3777.

3778.

3779.

3780.

3781.

3782. 3783. 3784.

3785. 3786.

3787.

3788.

3789.

3790. 3791.

Regulatory Hierarchies in Development.” Advances in Genetics, Vol. 27. Acad. Pr., New York. Scanga, S., Gupta, R., Nohyenk, D., and Manoukian, A. (2001). Regulation of the Wingless morphogen gradient by heparin-like glycosaminoglycans. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a149. Scanga, S., Manoukian, A., and Larsen, E. (1995). Timeand concentration-dependent response of the Drosophila antenna imaginal disc to Antennapedia. Dev. Biol. 169, 673–682. Schaefer, M., Petronczki, M., Dorner, D., Forte, M., and Knoblich, J.A. (2001). Heterotrimeric G proteins direct two modes of asymmetric cell division in the Drosophila nervous system. Cell 107, 183–194. [See also 2001 Cell 107, 125–128.] Schaefer, M., Shevchenko, A., Shevchenko, A., and Knoblich, J.A. (2000). A protein complex containing Inscuteable and the Gα-binding protein Pins orients asymmetric cell divisions in Drosophila. Curr. Biol. 10, 353– 362. ¨ Scheffzek, K., Ahmadian, M.R., Kabsch, W., Wiesmuller, L., Lautwein, A., Schmitz, F., and Wittinghofer, A. (1997). The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277, 333–338. Schejter, E.D., Segal, D., Glazer, L., and Shilo, B.-Z. (1986). Alternative 5 exons and tissue-speciﬁc expression of the Drosophila EGF receptor homolog transcripts. Cell 46, 1091–1101. Schejter, E.D. and Shilo, B.-Z. (1989). The Drosophila EGF receptor homolog (DER) gene is allelic to faint little ball, a locus essential for embryonic development. Cell 56, 1093– 1104. Schlake, T., Schorpp, M., and Boehm, T. (2000). Formation of regulator/target gene relationships during evolution. Gene 256, 29–34. Schlessinger, J. (1993). How receptor tyrosine kinases activate Ras. Trends Biochem. Sci. 18, 273–275. Schlessinger, J. (2000). Cell signaling by receptor tyrosine kinases. Cell 103, 211–225. Schlessinger, J., Plotnikov, A.N., Ibrahimi, O.A., Eliseenkova, A.V., Yeh, B.K., Yayon, A., Linhardt, R.J., and Mohammadi, M. (2000). Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Molec. Cell 6, 743–750. Schlessinger, J. and Ullrich, A. (1992). Growth factor signaling by receptor tyrosine kinases. Neuron 9, 383–391. Schmeichel, K.L. and Beckerle, M.C. (1994). The LIM domain is a modular protein-binding interface. Cell 79, 211– 219. Schmid, A.T., Tinley, T.L., and Yedvobnick, B. (1996). Transcription of the neurogenic gene mastermind during Drosophila development. J. Exp. Zool. 274, 207–220. Schmid, H. (1985). Transdetermination in the homeotic eye-antenna imaginal disc of Drosophila melanogaster. Dev. Biol. 107, 28–37. Schmid, H., Gendre, N., and Stocker, R.F. (1986). Surgical generation of supernumerary appendages for studying neuronal speciﬁcity in Drosophila melanogaster. Dev. Biol. 113, 160–173. Schmidt, K. (1994). A puzzle: how similar signals yield different effects. Science 266, 566–567. Schmidt-Ott, U., Gonz´alez-Gait´an, M., and Technau, G.M. (1995). Analysis of neural elements in head-mutant

410

3792.

3793.

3794.

3795.

3796.

3797.

3798.

3799. 3800.

3801.

3802.

3803.

3804.

3805.

3806.

3807.

3808.

3809.

REFERENCES

Drosophila embryos suggests segmental origin of the optic lobes. Roux’s Arch. Dev. Biol. 205, 31–44. Schmidt-Ott, U. and Technau, G.M. (1994). Fate-mapping in the procephalic region of the embryonic Drosophila head. Roux’s Arch. Dev. Biol. 203, 367–373. Schmucker, D., Clemens, J.C., Shu, H., Worby, C.A., Xiao, J., Muda, M., Dixon, J.E., and Zipursky, S.L. (2000). Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 101, 671– 684. Schneiderman, H.A. (1969). Control systems in insect development. In “Biology and the Physical Sciences,” (S. Devons, ed.). Columbia Univ. Pr.: New York, pp. 186–208. Schneiderman, H.A. (1976). New ways to probe pattern formation and determination in insects. In “Insect Development,” Symp. Roy. Entomol. Soc. Lond., Vol. 8 (P.A. Lawrence, ed.). Wiley: New York, pp. 3–34. Schneiderman, H.A. (1979). Pattern formation and determination in insects. In “Mechanisms of Cell Change,” (J. Ebert and T. Okada, eds.). Wiley: New York, pp. 243–272. Schneiderman, H.A. and Bryant, P.J. (1971). Genetic analysis of developmental mechanisms in Drosophila. Nature 234, 187–194. Schneitz, K., Spielmann, P., and Noll, M. (1993). Molecular genetics of aristaless, a prd-type homeo box gene involved in the morphogenesis of proximal and distal pattern elements in a subset of appendages in Drosophila. Genes Dev. 7, 114–129. Schnell, S. and Maini, P.K. (2000). Clock and induction model for somitogenesis. Dev. Dynamics 217, 415–420. Schnepp, B., Donaldson, T., Grumbling, G., Ostrowski, S., Schweitzer, R., Shilo, B.-Z., and Simcox, A. (1998). EGF domain swap converts a Drosophila EGF receptor activator into an inhibitor. Genes Dev. 12, 908–913. Schnepp, B., Grumbling, G., Donaldson, T., and Simcox, A. (1996). Vein is a novel component in the Drosophila epidermal growth factor receptor pathway with similarity to the neuregulins. Genes Dev. 10, 2302–2313. Schneuwly, S., Klemenz, R., and Gehring, W.J. (1987). Redesigning the body plan of Drosophila by ectopic expression of the homoeotic gene Antennapedia. Nature 325, 816–818. Schober, M., Schaefer, M., and Knoblich, J.A. (1999). Bazooka recruits Inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts. Nature 402, 548–551. Scholz, H., Deatrick, J., Klaes, A., and Kl¨ambt, C. (1993). Genetic dissection of pointed, a Drosophila gene encoding two ETS-related proteins. Genetics 135, 455–468. Schroeter, E.H., Kisslinger, J.A., and Kopan, R. (1998). Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393, 382–386. Schrons, H., Knust, E., and Campos-Ortega, J.A. (1992). The Enhancer of split Complex and adjacent genes in the 96F region of Drosophila melanogaster are required for segregation of neural and epidermal progenitor cells. Genetics 132, 481–503. Schubiger, G. (1968). Anlageplan, Determinationszustand und Transdeterminationsleistungen der m¨annlichen Vorderbeinscheibe von Drosophila melanogaster. W. Roux’ Arch. Entw.-Mech. Org. 160, 9–40. Schubiger, G. (1971). Regeneration, duplication and transdetermination in fragments of the leg disc of Drosophila melanogaster. Dev. Biol. 26, 277–295. Schubiger, G. (1974). Acquisition of differentiative com-

3810.

3811.

3812.

3813.

3814.

3815.

3816.

3817.

3818.

3819.

3820.

3821.

3822. 3823.

3824.

3825.

3826.

3827.

petence in the imaginal leg discs of Drosophila. W. Roux’ Arch. 174, 303–311. Schubiger, G. and Alpert, G.D. (1975). Regeneration and duplication in a temperature sensitive homeotic mutant of Drosophila melanogaster. Dev. Biol. 42, 292–304. Schubiger, G. and Schubiger, M. (1978). Distal transformation in Drosophila leg imaginal disc fragments. Dev. Biol. 67, 286–295. Schubiger, M. and Palka, J. (1985). Genetic suppression of putative guidepost cells: effect on establishment of nerve pathways in Drosophila wings. Dev. Biol. 108, 399–410. Schubiger, M. and Palka, J. (1987). Changing spatial patterns of DNA replication in the developing wing of Drosophila. Dev. Biol. 123, 145–153. Schubiger, M. and Truman, J.W. (2000). The RXR ortholog USP suppresses early metamorphic processes in Drosophila in the absence of ecdysteroids. Development 127, 1151–1159. Schulz, C. and Tautz, D. (1995). Zygotic caudal regulation by hunchback and its role in abdominal segment formation of the Drosophila embryo. Development 121, 1023– 1028. ¨ Schupbach, T. and Wieschaus, E. (1998). Probing for gene speciﬁcity in epithelial development. Int. J. Dev. Biol. 42, 249–255. ¨ Schupbach, T., Wieschaus, E., and N¨othiger, R. (1978). The embryonic organization of the genital disc studied in genetic mosaics of Drosophila melanogaster. W. Roux’s Arch. 185, 249–270. Schwartz, C., Locke, J., Nishida, C., and Kornberg, T.B. (1995). Analysis of cubitus interruptus regulation in Drosophila embryos and imaginal disks. Development 121, 1625–1635. Schwarz-Sommer, Z., Huijser, P., Nacken, W., Saedler, H., and Sommer, H. (1990). Genetic control of ﬂower development by homeotic genes in Antirrhinum majus. Science 250, 931–936. Schweisguth, F. (1995). Suppressor of Hairless is required for signal reception during lateral inhibition in the Drosophila pupal notum. Development 121, 1875–1884. Schweisguth, F. (1999). Dominant-negative mutation in the β2 and β6 proteasome subunit genes affect alternative cell fate decisions in the Drosophila sense organ lineage. Proc. Natl. Acad. Sci. USA 96, 11382–11386. Schweisguth, F. (2000). Cell polarity: Fixing cell polarity with Pins. Curr. Biol. 10, R265–R267. Schweisguth, F., Gho, M., and Lecourtois, M. (1996). Control of cell fate choices by lateral signaling in the adult peripheral nervous system of Drosophila melanogaster. Dev. Genet. 18, 28–39. Schweisguth, F. and Lecourtois, M. (1998). The activity of Drosophila Hairless is required in pupae but not in embryos to inhibit Notch signal transduction. Dev. Genes Evol. 208, 19–27. Schweisguth, F., Nero, P., and Posakony, J.W. (1994). The sequence similarity of the Drosophila Suppressor of Hairless protein to the integrase domain has no functional signiﬁcance in vivo. Dev. Biol. 166, 812–814. Schweisguth, F. and Posakony, J.W. (1992). Suppressor of Hairless, the Drosophila homolog of the mouse recombination signal-binding protein gene, controls sensory organ cell fates. Cell 69, 1199–1212. Schweisguth, F. and Posakony, J.W. (1994). Antagonistic activities of Suppressor of Hairless and Hairless control

REFERENCES

3828.

3829.

3830.

3831.

3832.

3833.

3834. 3835.

3836.

3837. 3838. 3839. 3840. 3841. 3842. 3843.

3844.

3845.

3846. 3847.

alternative cell fates in the Drosophila adult epidermis. Development 120, 1433–1441. Schweitzer, R., Howes, R., Smith, R., Shilo, B.-Z., and Freeman, M. (1995). Inhibition of Drosophila EGF receptor activation by the secreted protein Argos. Nature 376, 699– 702. Schweitzer, R., Shaharabany, M., Seger, R., and Shilo, B.-Z. (1995). Secreted Spitz triggers the DER signaling pathway and is a limiting component in embryonic ventral ectoderm determination. Genes Dev. 9, 1518–1529. Schweitzer, R. and Shilo, B.-Z. (1997). A thousand and one roles for the Drosophila EGF receptor. Trends Genet. 13, 191–196. Schweizer, L. and Basler, K. (1998). Drosophila ci D encodes a hybrid Pangolin/Cubitus interruptus protein that diverts the Wingless into the Hedgehog signaling pathway. Mechs. Dev. 78, 141–151. ¨ Schwenk, H. (1947). Untersuchungen uber die Entwicklung der Borsten bei Drosophila. Nachr. Akad. Wiss. G¨ottingen, Math-physik. Kl. IIb, Biol.-Physiol.-Chem. Abt. (1947), 14–16. Schwyter, D.H., Huang, J.-D., Dubnicoff, T., and Courey, A.J. (1995). The decapentaplegic core promoter region plays an integral role in the spatial control of transcription. Mol. Cell. Biol. 15, 3960–3968. Scott, J.D. and Pawson, T. (2000). Cell communication: the inside story. Sci. Am. 282 #6, 72–79. Scott, K., Brady, R., Jr., Cravchik, A., Morozov, P., Rzhetsky, A., Zuker, C., and Axel, R. (2001). A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila. Cell 104, 661–673. Scott, M.P. (1985). Molecules and puzzles from the antennapedia homoeotic gene complex of Drosophila. Trends Genet. 1, 74–80. Scott, M.P. (1986). More on the homeobox. BioEssays 5, 88–89. Scott, M.P. (1987). Complex loci of Drosophila. Annu. Rev. Biochem. 56, 195–227. Scott, M.P. (1992). Vertebrate homeobox gene nomenclature. Cell 71, 551–553. Scott, M.P. (1994). Intimations of a creature. Cell 79, 1121– 1124. Scott, M.P. (1997). A common language. Cold Spr. Harb. Symp. Quant. Biol. 62, 555–562. Scott, M.P. (1999). Hox proteins reach out round DNA. Nature 397, 649–651. Scott, M.P. and Carroll, S.B. (1987). The segmentation and homeotic gene network in early Drosophila development. Cell 51, 689–698. Scott, M.P. and Weiner, A.J. (1984). Structural relationships among genes that control development: sequence homology between the Antennapedia, Ultrabithorax, and fushi tarazu loci of Drosophila. Proc. Natl. Acad. Sci. USA 81, 4115–4119. Scowcroft, W.R., Green, M.M., and Latter, B.D.H. (1968). Dosage at the scute locus, and canalisation of anterior and posterior scutellar bristles in Drosophila melanogaster. Genetics 60, 373–388. Sears, R., Leone, G., DeGregori, J., and Nevins, J.R. (1999). Ras enhances Myc protein stability. Molec. Cell 3, 169–179. Sedkov, Y., Tillib, S., Mizrokhi, L., and Mazo, A. (1994). The bithorax complex is regulated by trithorax earlier during Drosophila embryogenesis than is the Antennapedia complex, correlating with a bithorax-like expression pat-

411

3848.

3849.

3850.

3851.

3852.

3853.

3854.

3855.

3856.

3857.

3858. 3859.

3860.

3861.

3862.

3863.

3864.

3865.

tern of distinct early trithorax transcripts. Development 120, 1907–1917. Sedlak, B.J., Manzo, R., and Stevens, M. (1984). Localized cell death in Drosophila imaginal wing disc epithelium caused by the mutation apterous-blot. Dev. Biol. 104, 489– 496. Seeling, J.M., Miller, J.R., Gil, R., Moon, R.T., White, R., and Virshup, D.M. (1999). Regulation of β-catenin signaling by the B56 subunit of protein phosphatase 2A. Science 283, 2089–2091. Segal, D. and Gelbert, W.M. (1985). Shortvein, a new component of the decapentaplegic gene complex in Drosophila melanogaster. Genetics 109, 119–143. Seimiya, M. and Gehring, W.J. (2000). The Drosophila homeobox gene optix is capable of inducing ectopic eyes by an eyeless-independent mechanism. Development 127, 1879–1886. Sekelsky, J.J., Newfeld, S.J., Raftery, L.A., Chartoff, E.H., and Gelbart, W.M. (1995). Genetic characterization and cloning of Mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster. Genetics 139, 1347–1358. Selleck, M.A.J. and Bronner-Fraser, M. (1995). Origins of the avian neural crest: the role of neural plate-epidermal interactions. Development 121, 525–538. Selleck, S.B. (2000). Proteoglycans and pattern formation: sugar biochemistry meets developmental genetics. Trends Genet. 16, 206–212. Selva, E.M., Hong, K., Baeg, G.H., Beverley, S.M., Turco, S.J., Perrimon, N., and Hacker, U. (2001). Dual role of the fringe connection gene in both heparin sulfate and fringedependent signalling events. Nature Cell Biol. 3, 809–815. Sengar, A.S., Wang, W., Bishay, J., Cohen, S., and Egan, S.E. (1999). The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15. EMBO J. 18, 1159–1171. Sepp, K.J. and Auld, V.J. (1999). Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster. Genetics 151, 1093–1101. Seraﬁni, T. (1999). Finding a partner in a crowd: neuronal diversity and synaptogenesis. Cell 98, 133–136. Serikaku, M.A. and O’Tousa, J.E. (1994). sine oculis is a homeobox gene required for Drosophila visual system development. Genetics 138, 1137–1150. Serrano, N., Brock, H.W., and Maschat, F. (1997). β3tubulin is directly repressed by the Engrailed protein in Drosophila. Development 124, 2527–2536. Serrano, N. and Maschat, F. (1998). Molecular mechanism of polyhomeotic activation by Engrailed. EMBO J. 17, 3704– 3713. Serrano, N. and O’Farrell, P.H. (1997). Limb morphogenesis: connections between patterning and growth. Curr. Biol. 7, R186–R195. Seugnet, L., Simpson, P., and Haenlin, M. (1997). Requirement for dynamin during Notch signaling in Drosophila neurogenesis. Dev. Biol. 192, 585–598. Sewalt, R.G.A.B., Gunster, M.J., van der Vlag, J., Satijn, D.P.E., and Otte, A.P. (1999). C-terminal binding protein is a transcriptional repressor that interacts with a speciﬁc class of vertebrate Polycomb proteins. Mol. Cell. Biol. 19, 777–787. Seybold, W.D. and Sullivan, D.T. (1978). Protein synthetic patterns during differentiation of imaginal discs in vitro. Dev. Biol. 65, 69–80.

412

3866. Seydoux, G. and Greenwald, I. (1989). Cell autonomy of lin-12 function in a cell fate decision in C. elegans. Cell 57, 1237–1245. 3867. Shanbhag, S.R., Singh, K., and Singh, R.N. (1992). Ultrastructure of the femoral chordotonal organs and their novel synaptic organization in the legs of Drosophila melanogaster Meigen (Diptera: Drosophilidae). Int. J. Insect Morphol. & Embryol. 21, 311–322. 3868. Shanower, G., Muller, M., Blanton, J., Gyurkovics, H., and Schedl, P. (2001). grappa: a new trithorax/Polycomb-group gene involved in telomere silencing. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a98. 3869. Shao, Z., Raible, F., Mollaaghababa, R., Guyon, J.R., Wu, C.t., Bender, W., and Kingston, R.E. (1999). Stabilization of chromatin structure by PRC1, a Polycomb complex. Cell 98, 37–46. 3870. Shapiro, L. and Colman, D.R. (1999). The diversity of cadherins and implications for a synaptic adhesive code in the CNS. Neuron 23, 427–430. 3871. Sharkey, M., Graba, Y., and Scott, M.P. (1997). Hox genes in evolution: protein surfaces and paralog groups. Trends Genet. 13, 145–151. 3872. Sharp, E.J., Martin, E.C., and Adler, P.N. (1994). Directed overexpression of Suppressor 2 of zeste and Posterior Sex Combs results in bristle abnormalities in Drosophila melanogaster. Dev. Biol. 161, 379–392. 3873. Sharpe, C., Lawrence, N., and Martinez Arias, A. (2001). Wnt signalling: a theme with nuclear variations. BioEssays 23, 311–318. 3874. Sharrocks, A.D., Yang, S.-H., and Galanis, A. (2000). Docking domains and substrate speciﬁcity determination for MAP kinases. Trends Biochem. 25, 448–453. 3875. Shashidhara, L.S., Agrawal, N., Bajpai, R., Bharathi, V., and Sinha, P. (1999). Negative regulation of dorsoventral signaling by the homeotic gene Ultrabithorax during haltere development in Drosophila. Dev. Biol. 212, 491–502. 3876. Shaw, G. (1996). The pleckstrin homology domain: an intriguing multifunctional protein module. BioEssays 18, 35–46. 3877. Shaw, S.R. and Meinertzhagen, I.A. (1986). Evolutionary progression at synaptic connections made by identiﬁed homologous neurones. Proc. Natl. Acad. Sci. USA 83, 7961– 7965. 3878. Shaw, V.K. and Bryant, P.J. (1975). Intercalary leg regeneration in the large milkweed bug Oncopeltus fasciatus. Dev. Biol. 45, 187–191. 3879. Shaywitz, A.J. and Greenberg, M.E. (1999). CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu. Rev. Biochem. 68, 821–861. 3880. Shearn, A. (1977). Mutational dissection of imaginal disc development in Drosophila melanogaster. Amer. Zool. 17, 585–594. 3881. Shearn, A. (1978). Mutational dissection of imaginal disc development. In “The Genetics and Biology of Drosophila,” Vol. 2c (M. Ashburner and T.R.F. Wright, eds.). Acad. Pr.: New York, pp. 443–510. 3882. Shearn, A. (1980). What is the normal function of genes which give rise to homeotic mutations? In “Development and Neurobiology of Drosophila,” (O. Siddiqi, P. Babu, L.M. Hall, and J.C. Hall, eds.). Plenum: New York, pp. 155– 162. 3883. Shearn, A. (1985). Analysis of transdetermination. In “Comprehensive Insect Physiology, Biochemistry, and

REFERENCES

3884.

3885.

3886.

3887.

3888.

3889.

3890.

3891.

3892.

3893.

3894.

3895.

3896.

3897. 3898.

3899.

3900.

3901.

Pharmacology,” Vol. 2 (G.A. Kerkut and L.I. Gilbert, eds.). Pergamon Pr.: Oxford, pp. 183–199. Shearn, A. (1989). The ash-1, ash-2 and trithorax genes of Drosophila melanogaster are functionally related. Genetics 121, 517–525. Shearn, A., Davis, K.T., and Hersperger, E. (1978). Transdetermination of Drosophila imaginal discs cultured in vitro. Dev. Biol. 65, 536–540. Shearn, A. and Garen, A. (1974). Genetic control of imaginal disc development in Drosophila. Proc. Natl. Acad. Sci. USA 71, 1393–1397. Shearn, A., Martin, A., Davis, K., and Hersperger, E. (1984). Genetic analysis of transdetermination in Drosophila. I. The effects of varying growth parameters using a temperature-sensitive mutation. Dev. Biol. 106, 135–146. Shearn, A., Rice, T., Garen, A., and Gehring, W. (1971). Imaginal disc abnormalities in lethal mutants of Drosophila. Proc. Natl. Acad. Sci. USA 68, 2594–2598. Sheldahl, L.C., Park, M., Malbon, C.C., and Moon, R.T. (1999). Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner. Curr. Biol. 9, 695–698. Sheldon, B.L. (1968). Studies on the scutellar bristles of Drosophila melanogaster. I. Basic variability, some temperature and culture effects, and responses to short-term selection in the Oregon-RC strain. Aust. J. Biol. Sci. 21, 721–740. Shellenbarger, D.L. and Mohler, J.D. (1978). Temperaturesensitive periods and autonomy of pleiotropic effects of l(1)Nts1 , a conditional Notch lethal in Drosophila. Dev. Biol. 62, 432–446. Shen, C.-P., Jan, L.Y., and Jan, Y.N. (1997). Miranda is required for the asymmetric localization of Prospero during mitosis in Drosophila. Cell 90, 449–458. Shen, C.-P., Knoblich, J.A., Chan, Y.-M., Jiang, M.-M., Jan, L.Y., and Jan, Y.N. (1998). Miranda as a multidomain adapter linking apically localized Inscuteable and basally localized Staufen and Prospero during asymmetric cell division in Drosophila. Genes Dev. 12, 1837–1846. Shen, W. and Mardon, G. (1997). Ectopic eye development in Drosophila induced by directed dachshund expression. Development 124, 45–52. Sheng, G., Thouvenot, E., Schmucker, D., Wilson, D.S., and Desplan, C. (1997). Direct regulation of rhodopsin 1 by Pax-6/eyeless in Drosophila: evidence for a conserved function in photoreceptors. Genes Dev. 11, 1122–1131. Shepard, S.B., Broverman, S.A., and Muskavitch, M.A.T. (1989). A tripartite interaction among alleles of Notch, Delta, and Enhancer of split during imaginal development of Drosophila melanogaster. Genetics 122, 429–438. Shi, Y. (2001). Structural insights on Smad function in TGFβ signaling. BioEssays 23, 223–232. Shi, Y., Wang, Y.-F., Jayaraman, L., Yang, H., Massagu´e, J., and Pavletich, N.P. (1998). Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-β signaling. Cell 94, 585–594. Shields, J.M., Pruitt, K., McFall, A., Shaub, A., and Der, C.J. (2000). Understanding Ras: “It ain’t over ’til it’s over”. Trends Cell Biol. 10, 147–154. Shilo, B.-Z. and Raz, E. (1991). Developmental control by the Drosophila EGF receptor homolog DER. Trends Genet. 7, 388–392. Shimell, M.J., Ferguson, E.L., Childs, S.R., and O’Connor, M.B. (1991). The Drosophila dorsal-ventral patterning

REFERENCES

3902.

3903.

3904.

3905.

3906.

3907. 3908.

3909. 3910.

3911. 3912.

3913.

3914.

3915.

3916.

3917.

3918.

3919.

gene tolloid is related to human bone morphogenetic protein 1. Cell 67, 469–481. Shimell, M.J., Simon, J., Bender, W., and O’Connor, M.B. (1994). Enhancer point mutation results in a homeotic transformation in Drosophila. Science 264, 968–971. Shine, I. and Wrobel, S. (1976). “Thomas Hunt Morgan, Pioneer of Genetics.” Kentucky Univ. Pr., Lexington, Kentucky. Shiomi, K., Takeichi, M., Nishida, Y., Nishi, Y., and Uemura, T. (1994). Alternative cell fate choice induced by low-level expression of a regulator of protein phosphatase 2A in the Drosophila peripheral nervous system. Development 120, 1591–1599. Shirras, A.D. and Couso, J.P. (1996). Cell fates in the adult abdomen of Drosophila are determined by wingless during pupal development. Dev. Biol. 175, 24–36. Shishido, E., Higashijima, S.-i., Emori, Y., and Saigo, K. (1993). Two FGF-receptor homologues of Drosophila: one is expressed in mesodermal primordium in early embryos. Development 117, 751–761. Shore, P. and Sharrocks, A.D. (1995). The MADS-box family of transcription factors. Eur. J. Biochem. 229, 1–13. Shoresh, M., Orgad, S., Shmueli, O., Werczberger, R., Gelbaum, D., Abiri, S., and Segal, D. (1998). Overexpression Beadex mutations and loss-of-function heldup-a mutations in Drosophila affect the 3 regulatory and coding components, respectively, of the Dlmo Gene. Genetics 150, 283–299. Shostak, S. (1998). “Death of Life: The Legacy of Molecular Biology.” MacMillan, London. Shtutman, M., Zhurinsky, J., Simcha, I., Albanese, C., D’Amico, M., Pestell, R., and Ben-Ze’ev, A. (1999). The cyclin D1 gene is a target of the β-catenin/LEF-1 pathway. Proc. Natl. Acad. Sci. USA 96, 5522–5527. Shubin, N., Tabin, C., and Carroll, S. (1997). Fossils, genes and the evolution of animal limbs. Nature 388, 639–648. Shulman, J.M., Perrimon, N., and Axelrod, J.D. (1998). Frizzled signaling and the developmental control of cell polarity. Trends Genet. 14, 452–458. [See also 2001 references: Cell 106, 355–366; Curr. Biol. 11, R506–R509; Development 128, 3209–3220; Genes Dev. 15, 1182–1187.] Shupliakov, O., L¨ow, P., Grabs, D., Gad, H., Chen, H., David, C., Takei, K., De Camilli, P., and Brodin, L. (1997). Synaptic vesicle endocytosis impaired by disruption of dynaminSH3 domain interactions. Science 276, 259–263. Sibatani, A. (1977). Possible interaction of positional information and its interpretation in development: a model. Australian Biochem. Soc. 10, 74. Sibatani, A. (1981). The Polar Co-ordinate Model for pattern regulation in epimorphic ﬁelds: a critical appraisal. J. Theor. Biol. 93, 433–489. Sibatani, A. (1983). A plausible molecular interpretation of the polar coordinate model with a centripetal degeneracy of distinctive ﬁeld values. J. Theor. Biol. 103, 421–428. Sibbons, J.P., Shandala, T., and Saint, R. (2001). Characterisation of the Drosophila dead ringer gene in eye development. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a272. Siddiqi, O. and Rodrigues, V. (1980). Genetic analysis of a complex chemoreceptor. In “Development and Neurobiology of Drosophila,” (O. Siddiqi, P. Babu, L.M. Hall, and J.C. Hall, eds.). Plenum: New York, pp. 347–359. Siegfried, E. (1999). Role of Drosophila Wingless signaling in cell fate determination. In “Cell Lineage and Fate

413

3920.

3921.

3922.

3923.

3924.

3925.

3926.

3927.

3928.

3929.

3930.

3931.

3932.

3933.

3934.

3935.

Determination,” (S.A. Moody, ed.). Acad. Pr.: New York, pp. 249–271. [See also 2001 references concerning Wingless secretion (BioEssays 23, 869–872; Cell 105, 197–207, 209–219, and 613–624; Curr.Biol. 11, R638–R639); binding (Dev. Biol. 235, 467–475; Nature 412, 86–90); transduction (Cell 105, 391–402 and 563–566; Curr.Biol. 11, R524–R526; Dev.Biol. 235, 303–313 and 433–448); and other Wnts (Dev. Biol. 232, 339–350; Mechs. Dev. 103, 117–120).] Siegfried, E., Ambrosio, L., and Perrimon, N. (1990). Serine/threonine protein kinases in Drosophila. Trends Genet. 6, 357–362. Siegfried, E., Chou, T.-B., and Perrimon, N. (1992). wingless signaling acts through zeste-white 3, the Drosophila homolog of glycogen synthase kinase-3, to regulate engrailed and establish cell fate. Cell 71, 1167–1179. Siegfried, E., Perkins, L.A., Capaci, T.M., and Perrimon, N. (1990). Putative protein kinase product of the Drosophila segment-polarity gene zeste-white3. Nature 345, 825–829. Siegfried, E. and Perrimon, N. (1994). Drosophila wingless: a paradigm for the function and mechanism of Wnt signaling. BioEssays 16, 395–404. Siegfried, E., Wilder, E.L., and Perrimon, N. (1994). Components of wingless signalling in Drosophila. Nature 367, 76–80. Siegler, M.V.S. and Jia, X.X. (1999). Engrailed negatively regulates the expression of cell adhesion molecules Connectin and Neuroglian in embryonic Drosophila nervous system. Neuron 22, 265–276. Sigrist, S., Jacobs, H., Stratmann, R., and Lehner, C.F. (1995). Exit from mitosis is regulated by Drosophila ﬁzzy and the sequential destruction of cyclins A, B and B3. EMBO J. 14, 4827–4838. Sigrist, S.J. and Lehner, C.F. (1997). Drosophila ﬁzzyrelated down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. Cell 90, 671–681. Simcox, A. (1997). Differential requirement for EGF-like ligands in Drosophila wing development. Mechs. Dev. 62, 41–50. Simcox, A.A., Grumbling, G., Schnepp, B., BenningtonMathias, C., Hersperger, E., and Shearn, A. (1996). Molecular, phenotypic, and expression analysis of vein, a gene required for growth of the Drosophila wing disc. Dev. Biol. 177, 475–489. Simcox, A.A., Hersperger, E., Shearn, A., Whittle, J.R.S., and Cohen, S.M. (1991). Establishment of imaginal discs and histoblast nests in Drosophila. Mechs. Dev. 34, 11–20. Simcox, A.A., Wurst, G., Hersperger, E., and Shearn, A. (1987). The defective dorsal discs gene of Drosophila is required for the growth of speciﬁc imaginal discs. Dev. Biol. 122, 559–567. (N.B.: The “ddd” gene studied here was renamed “vein”.) Simin, K., Bates, E.A., Horner, M.A., and Letsou, A. (1998). Genetic analysis of Punt, a type II Dpp receptor that functions throughout the Drosophila melanogaster life cycle. Genetics 148, 801–813. Simmonds, A., Hughes, S., Tse, J., Cocquyt, S., and Bell, J. (1997). The effect of dominant vestigial alleles upon vestigial-mediated wing patterning during development of Drosophila melanogaster. Mechs. Dev. 67, 17–33. Simmonds, A.J. and Bell, J.B. (1998). A genetic and molecular analysis of an invected Dominant mutation in Drosophila melanogaster. Genome 41, 381–390. Simmonds, A.J., Brook, W.J., Cohen, S.M., and Bell, J.B.

414

3936.

3937.

3938.

3939.

3940.

3941.

3942. 3943.

3944.

3945.

3946. 3947.

3948.

3949. 3950.

3951. 3952.

3953. 3954.

REFERENCES

(1995). Distinguishable functions for engrailed and invected in anterior-posterior patterning in the Drosophila wing. Nature 376, 424–427. Simmonds, A.J., Liu, X., Soanes, K.H., Krause, H.M., Irvine, K.D., and Bell, J.B. (1998). Molecular interactions between Vestigial and Scalloped promote wing formation in Drosophila. Genes Dev. 12, 3815–3820. [See also 2001 Development 128, 3295–3305.] Simmons, D.L. (1993). Dissecting the modes of interactions amongst cell adhesion molecules. Development 1993 Suppl., 193–203. Simon, J. (1995). Locking in stable states of gene expression: transcriptional control during Drosophila development. Curr. Opin. Cell Biol. 7, 376–385. Simon, J., Bornemann, D., Lunde, K., and Schwartz, C. (1995). The extra sex combs product contains WD40 repeats and its time of action implies a role distinct from other Polycomb group products. Mechs. Dev. 53, 197–208. [See also 2001 Genes Dev. 15, 2509–2514.] Simon, J., Peifer, M., Bender, W., and O’Connor, M. (1990). Regulatory elements of the bithorax complex that control expression along the anterior-posterior axis. EMBO J. 9, 3945–3956. Simon, M.A. (1994). Signal transduction during the development of the Drosophila R7 photoreceptor. Dev. Biol. 166, 431–442. Simon, M.A. (2000). Receptor tyrosine kinases: speciﬁc outcomes from general signals. Cell 103, 13–15. Simon, M.A., Bowtell, D.D.L., Dodson, G.S., Laverty, T.R., and Rubin, G.M. (1991). Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the Sevenless protein tyrosine kinase. Cell 67, 701–716. Simon, M.A., Dodson, G.S., and Rubin, G.M. (1993). An SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to Sevenless and Sos proteins in vitro. Cell 73, 169–177. Simonelig, M., Elliott, K., Mitchelson, A., and O’Hare, K. (1996). Interallelic complementation at the suppressor of forked locus of Drosophila reveals complementation between Suppressor of forked proteins mutated in different regions. Genetics 142, 1225–1235. Simons, K. and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387, 569–572. Simpson, P. (1976). Analysis of the compartments of the wing of Drosophila melanogaster mosaic for a temperature-sensitive mutation that reduces mitotic rate. Dev. Biol. 54, 100–115. Simpson, P. (1979). Parameters of cell competition in the compartments of the wing disc of Drosophila. Dev. Biol. 69, 182–193. Simpson, P. (1981). Growth and cell competition in Drosophila. J. Embryol. Exp. Morph. 65 (Suppl.), 77–88. Simpson, P. (1990). Lateral inhibition and the development of the sensory bristles of the adult peripheral nervous system of Drosophila. Development 109, 509– 519. Simpson, P. (1990). Notch and the choice of cell fate in Drosophila neuroepithelium. Trends Genet. 6, 343–345. Simpson, P. (1993). Flipping fruit-ﬂies: a powerful new technique for generating Drosophila mosaics. Trends Genet. 9, 227–228. Simpson, P. (1996). Drosophila development: A prepattern for sensory organs. Curr. Biol. 6, 948–950. Simpson, P. (1997). Notch signalling in development: on

3955.

3956.

3957.

3958.

3959.

3960.

3961.

3962.

3963.

3964.

3965.

3966.

3967.

3968.

3969.

3970.

equivalence groups and asymmetric developmental potential. Curr. Op. Gen. Dev. 7, 537–542. Simpson, P., Berreur, P., and Berreur-Bonnenfant, J. (1980). The initiation of pupariation in Drosophila: dependence on growth of the imaginal discs. J. Embryol. Exp. Morph. 57, 155–165. Simpson, P. and Carteret, C. (1989). A study of shaggy reveals spatial domains of expression of achaete-scute alleles on the thorax of Drosophila. Development 106, 57–66. Simpson, P. and Carteret, C. (1990). Proneural clusters: equivalence groups in the epithelium of Drosophila. Development 110, 927–932. Simpson, P., El Messal, M., Moscoso del Prado, J., and Ripoll, P. (1988). Stripes of positional homologies across the wing blade of Drosophila melanogaster. Development 103, 391–401. Simpson, P. and Grau, Y. (1987). The segment polarity gene costal-2 in Drosophila. II. The origin of imaginal pattern duplications. Dev. Biol. 122, 201–209. Simpson, P., Lawrence, P.A., and Maschat, F. (1981). Clonal analysis of two wing-scalloping mutants of Drosophila. Dev. Biol. 84, 206–211. Simpson, P. and Morata, G. (1980). The control of growth in the imaginal discs of Drosophila. In “Development and Neurobiology of Drosophila,” (O. Siddiqi, P. Babu, L.M. Hall, and J.C. Hall, eds.). Plenum: New York, pp. 129–139. Simpson, P. and Morata, G. (1981). Differential mitotic rates and patterns of growth in compartments in the Drosophila wing. Dev. Biol. 85, 299–308. Simpson, P., Ruel, L., Heitzler, P., and Bourouis, M. (1993). A dual role for the protein kinase shaggy in the repression of achaete-scute. Development 1993 Suppl., 29–39. Simpson, P. and Schneiderman, H.A. (1975). Isolation of temperature sensitive mutations blocking clone development in Drosophila melanogaster, and the effects of a temperature sensitive cell lethal mutation on pattern formation in imaginal discs. Wilhelm Roux’s Arch. 178, 247–275. Simpson, P. and Schneiderman, H.A. (1976). A temperature sensitive mutation that reduces mitotic rate in Drosophila melanogaster. W. Roux’s Arch. 179, 215–236. Simpson, P., Woehl, R., and Usui, K. (1999). The development and evolution of bristle patterns in Diptera. Development 126, 1349–1364. Sinclair, A.H., Berta, P., Palmer, M.S., Hawkins, J.R., Grifﬁths, B.L., Smith, M.J., Foster, J.W., Frischauf, A.-M., LovellBadge, R., and Goodfellow, P.N. (1990). A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346, 240–244. Sinclair, D.A.R., Clegg, N.J., Antonchuk, J., Milne, T.A., Stankunas, K., Ruse, C., Grigliatti, T.A., Kassis, J.A., and Brock, H.W. (1998). Enhancer of Polycomb is a suppressor of position-effect variegation in Drosophila melanogaster. Genetics 148, 211–220. Sinclair, D.A.R., Grigliatti, T.A., and Kaufman, T.C. (1984). Effects of a temperature-sensitive Minute mutation on gene expression in Drosophila melanogaster. Genet. Res., Camb. 43, 257–275. Sinclair, D.A.R., Milne, T.A., Hodgson, J.W., Shellard, J., Salinas, C.A., Kyba, M., Randazzo, F., and Brock, H.W. (1998). The Additional sex combs gene of Drosophila encodes a chromatin protein that binds to shared and unique Polycomb group sites on polytene chromosomes. Development 125, 1207–1216.

REFERENCES

3971. Singer, M.A., Hortsch, M., Goodman, C.S., and Bentley, M. (1992). Annulin, a protein expressed at limb segment boundaries in the grasshopper embryo, is homologous to protein cross-linking transglutaminases. Dev. Biol. 154, 143–159. 3972. Singer, M.A., Penton, A., Twombly, V., Hoffmann, F.M., and Gelbart, W.M. (1997). Signaling through both type I DPP receptors is required for anterior-posterior patterning of the entire Drosophila wing. Development 124, 79–89. 3973. Singh, A., Kango-Singh, M., and Sun, Y.H. (2001). teashirt collaborates with Wg to suppress ventral eye development. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a202. 3974. Singson, A., Leviten, M.W., Bang, A.G., Hua, X.H., and Posakony, J.W. (1994). Direct downstream targets of proneural activators in the imaginal disc include genes involved in lateral inhibitory signaling. Genes Dev. 8, 2058– 2071. 3975. Sipos, L., Mih´aly, J., Karch, F., Schedl, P., Gausz, J., and Gyurkovics, H. (1998). Transvection in the Drosophila AbdB domain: extensive upstream sequences are involved in anchoring distant cis-regulatory regions to the promoter. Genetics 149, 1031–1050. 3976. Sisson, J.C., Ho, K.S., Suyama, K., and Scott, M.P. (1997). Costal2, a novel kinesin-related protein in the Hedgehog signaling pathway. Cell 90, 235–245. 3977. Sivasankaran, R., Calleja, M., Morata, G., and Basler, K. (2000). The Wingless target gene Dfz3 encodes a new member of the Drosophila Frizzled family. Mechs. Dev. 91, 427–431. ¨ 3978. Sivasankaran, R., Vigano, M.A., Muller, B., Affolter, M., and Basler, K. (2000). Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker. EMBO J. 19, 6162–6172. 3979. Skaer, H. (1998). Who pulls the string to pattern cell division in Drosophila? Trends Genet. 14, 337–339. 3980. Skaer, N. and Simpson, P. (2000). Genetic analysis of bristle loss in hybrids between Drosophila melanogaster and D. simulans provides evidence for divergence of cisregulatory sequences in the achaete-scute gene complex. Dev. Biol. 221, 148–167. 3981. Skeath, J.B. (1999). At the nexus between pattern formation and cell-type speciﬁcation: the generation of individual neuroblast fates in the Drosophila embryonic central nervous system. BioEssays 21, 922–931. 3982. Skeath, J.B. and Carroll, S.B. (1991). Regulation of achaetescute gene expression and sensory organ pattern formation in the Drosophila wing. Genes Dev. 5, 984–995. 3983. Skeath, J.B. and Carroll, S.B. (1992). Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo. Development 114, 939– 946. 3984. Skeath, J.B. and Carroll, S.B. (1994). The achaete-scute complex: generation of cellular pattern and fate within the Drosophila nervous system. FASEB J. 8, 714–721. 3985. Skeath, J.B. and Doe, C.Q. (1998). Sanpodo and Notch act in opposition to Numb to distinguish sibling neuron fates in the Drosophila CNS. Development 125, 1857–1865. 3986. Skeath, J.B., Panganiban, G., Selegue, J., and Carroll, S.B. (1992). Gene regulation in two dimensions: the proneural achaete and scute genes are controlled by combinations of axis-patterning genes through a common intergenic control region. Genes Dev. 6, 2606–2619. 3987. Skripsky, T. and Lucchesi, J.C. (1982). Intersexuality result-

415

3988. 3989. 3990.

3991.

3992.

3993.

3994.

3995.

3996.

3997.

3998.

3999. 4000. 4001. 4002.

4003.

4004.

4005. 4006.

ing from the interaction of sex-speciﬁc lethal mutations in Drosophila melanogaster. Dev. Biol. 94, 153–162. Slack, J.M.W. (1987). Morphogenetic gradients–past and present. Trends Bioch. Sci. 12, 200–204. Slack, J.M.W. (1994). How to make the gradient. Nature 371, 477–478. Sliter, T.J. (1989). Imaginal disc-autonomous expression of a defect in sensory bristle patterning caused by the lethal(3)ecdysoneless 1 (l(3)ecd 1 ) mutation of Drosophila melanogaster. Development 106, 347–354. Slusarski, D.C., Motzny, C.K., and Holmgren, R. (1995). Mutations that alter the timing and pattern of cubitus interruptus gene expression in Drosophila melanogaster. Genetics 139, 229–240. Small, S., Arnosti, D.N., and Levine, M. (1993). Spacing ensures autonomous expression of different stripe enhancers in the even-skipped promoter. Development 119, 767–772. Small, S., Blair, A., and Levine, M. (1996). Regulation of two pair-rule stripes by a single enhancer in the Drosophila embryo. Dev. Biol. 175, 314–324. Smalley, M.J., Sara, E., Paterson, H., Naylor, S., Cook, D., Jayatilake, H., Fryer, L.G., Hutchinson, L., Fry, M.J., and Dale, T.C. (1999). Interaction of Axin and Dvl-2 proteins regulates Dvl-2-stimulated TCF-dependent transcription. EMBO J. 18, 2823–2835. Smith, A.V. and Orr-Weaver, T.L. (1991). The regulation of the cell cycle during Drosophila embryogenesis: the transition to polyteny. Development 112, 997–1008. Smith, C.W.J. and Valc´arcel, J. (2000). Alternative premRNA splicing: the logic of combinatorial control. Trends Biochem. Sci. 25, 381–388. Smith, E., Pannuti, A., Allis, C.D., and Lucchesi, J.C. (2001). Mapping of MSL proteins and H4 acetylated at lysine 16 on dosage compensation genes. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a90. Smith, E.R., Belote, J.M., Schiltz, R.L., Yang, X.-J., Moore, P.A., Berger, S.L., Nakatani, Y., and Allis, C.D. (1998). Cloning of Drosophila GCN5: conserved features among metazoan GCN5 family members. Nucl. Acids Res. 26, 2948–2954. Smith, J. (1996). How to tell a cell where it is. Nature 381, 367–368. Smith, J. (1997). Brachyury and the T-box genes. Curr. Opin. Gen. Dev. 7, 474–480. Smith, J. (1999). T-box genes: what they do and how they do it. Trends Genet. 15, 154–158. Smith, R.K., Carroll, P.M., Allard, J.D., and Simon, M.A. (2002). MASK, a large ankyrin repeat and KH domaincontaining protein involved in Drosophila receptor tyrosine kinase signaling. Development 129, 71–82. Smith, S.T. and Jaynes, J.B. (1996). A conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2- and mshclass homeoproteins, mediates active transcriptional repression in vivo. Development 122, 3141–3150. Smith, T.F., Gaitatzes, C., Saxena, K., and Neer, E.J. (1999). The WD repeat: a common architecture for diverse functions. Trends Biochem. Sci. 24, 181–185. Smolen, P., Baxter, D.A., and Byrne, J.H. (2000). Mathematical modeling of gene networks. Neuron 26, 567–580. Smolik-Utlaut, S.M. (1990). Dosage requirements of Ultrabithorax and bithoraxoid in the determination of segment identity in Drosophila melanogaster. Genetics 124, 357–366.

416

4007. Smoller, D., Friedel, C., Schmid, A., Bettler, D., Lam, L., and Yedvobnick, B. (1990). The Drosophila neurogenic locus mastermind encodes a nuclear protein unusually rich in amino acid homopolymers. Genes Dev. 4, 1688– 1700. 4008. Snodgrass, R.E. (1935). “Principles of Insect Morphology.” McGraw-Hill, New York. 4009. Snodgrass, R.E. (1958). Evolution of arthropod mechanisms. Smithsonian Misc. Coll. 138 #2, i-77. 4010. Snow, M.H.L. and Tam, P.P.L. (1980). Timing in embryological development. Nature 286, 107. [See also 2001 Cell 106, 133–136.] 4011. Soll, D.R. (1983). A new method for examining the complexity and relationships of “timers” in developing systems. Dev. Biol. 95, 73–91. 4012. Sondek, J., Bohm, A., Lambright, D.G., Hamm, H.E., and Sigler, P.B. (1996). Crystal structure of a GA protein βγ ˚ resolution. Nature 379, 369–374. dimer at 2.1 A 4013. Sondhi, K.C. (1962). The evolution of a pattern. Evolution 16, 186–191. 4014. Sondhi, K.C. (1963). The biological foundations of animal patterns. Quart. Rev. Biol. 38, 289–327. 4015. Sondhi, K.C. (1965). Genetic control of an anteroposterior gradient and its bearing on structural orientation in Drosophila. Genetics 51, 653–657. 4016. Sondhi, K.C. (1970). Dynamics of pattern formation. Genetica 41, 111–118. 4017. Song, Y., Chung, S., and Kunes, S. (2000). Combgap relays Wingless signal reception to the determination of cortical cell fate in the Drosophila visual system. Molec. Cell 6, 1143–1154. 4018. Song, Z., McCall, K., and Steller, H. (1997). DCP-1, a Drosophila cell death protease essential for development. Science 275, 536–540. 4019. Songyang, Z., Fanning, A.S., Fu, C., Xu, J., Marfatia, S.M., Chishti, A.H., Crompton, A., Chan, A.C., Anderson, J.M., and Cantley, L.C. (1997). Recognition of unique carboxylterminal motifs by distinct PDZ domains. Science 275, 73– 77. 4020. Songyang, Z., Shoelson, S.E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W.G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R.J., Neel, B.G., Birge, R.B., Fajardo, J.E., Chou, M.M., Hanafusa, H., Schaffhausen, B., and Cantley, L.C. (1993). SH2 domains recognize speciﬁc phosphopeptide sequences. Cell 72, 767–778. 4021. Sonnenblick, B.P. (1950). The early embryology of Drosophila melanogaster. In “Biology of Drosophila,” (M. Demerec, ed.). Hafner: New York, pp. 62–167. 4022. Sonnenfeld, M., Ward, M., Nystrom, G., Mosher, J., Stahl, S., and Crews, S. (1997). The Drosophila tango gene encodes a bHLH-PAS protein that is orthologous to mammalian Arnt and controls CNS midline and tracheal development. Development 124, 4571–4582. 4023. Sonnenfeld, M.J. and Jacobs, J.R. (1994). Mesectodermal cell fate analysis in Drosophila midline mutants. Mechs. Dev. 46, 3–13. 4024. Sotillos, S. and Campuzano, S. (2000). DRacGAP, a novel Drosophila gene, inhibits EGFR/Ras signalling in the developing imaginal wing disc. Development 127, 5427– 5438. 4025. Sotillos, S., Roch, F., and Campuzano, S. (1997). The metalloprotease-disintegrin Kuzbanian participates in Notch activation during growth and patterning of Drosophila imaginal discs. Development 124, 4769–4779.

REFERENCES

4026. Soto, M.C., Chou, T.-B., and Bender, W. (1995). Comparison of germline mosaics of genes in the Polycomb Group of Drosophila melanogaster. Genetics 140, 231–243. 4027. Spana, E.P. and Doe, C.Q. (1996). Numb antagonizes Notch signaling to specify sibling neuron cell fates. Neuron 17, 21–26. 4028. Spana, E.P., Kopczynski, C., Goodman, C.S., and Doe, C.Q. (1995). Asymmetric localization of Numb autonomously determines sibling neuron identity in the Drosophila CNS. Development 121, 3489–3494. 4029. Speicher, S.A., Thomas, U., Hinz, U., and Knust, E. (1994). The Serrate locus of Drosophila and its role in morphogenesis of the wing imaginal discs: control of cell proliferation. Development 120, 535–544. 4030. Spemann, H. (1938). “Embryonic Development and Induction.” Yale Univ. Pr., New Haven. 4031. Spencer, E., Jiang, J., and Chen, Z.J. (1999). Signal-induced ubiquitination of IκBα by the F-box protein Slimb/β-TrCP. Genes Dev. 13, 284–294. 4032. Spencer, F.A. (1984). “The Decapentaplegic Gene Complex and Adult Pattern Formation in Drosophila.” Ph.D. Dissertation, Harvard U., 4033. Spencer, F.A., Hoffmann, F.M., and Gelbart, W.M. (1982). Decapentaplegic: A gene complex affecting morphogenesis in Drosophila melanogaster. Cell 28, 451–461. 4034. Spencer, S.A. and Cagan, R. (2001). E(Elp)24D, an EGFreceptor inhibitor, is essential for R8 patterning. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a194. 4035. Spencer, S.A., Powell, P.A., Miller, D.T., and Cagan, R.L. (1998). Regulation of EGF receptor signaling establishes pattern across the developing Drosophila retina. Development 125, 4777–4790. 4036. Spickett, S.G. (1963). Genetic and developmental studies of a quantitative character. Nature 199, 870–873. 4037. Spiegelman, S. (1945). Physiological competition as a regulatory mechanism in morphogenesis. Q. Rev. Biol. 20, 121–146. 4038. Spiegelman, V.S., Slaga, T.J., Pagano, M., Minamoto, T., Ronai, Z., and Fuchs, S.Y. (2000). Wnt/β-catenin signaling induces the expression and activity of βTrCP ubiquitin ligase receptor. Molec. Cell 5, 877–882. 4039. Spieth, H.T. (1974). Courtship behavior in Drosophila. Annu. Rev. Entomol. 19, 385–405. 4040. Spink, K.E., Polakis, P., and Weis, W.I. (2000). Structural basis of the Axin-adenomatous polyposis coli interaction. EMBO J. 19, 2270–2279. 4041. Spitzer, N.C. and Sejnowski, T.J. (1997). Biological information processing: bits of progress. Science 277, 1060– 1061. 4042. Sprague, G.F., Jr. (1990). Combinatorial associations of regulatory proteins and the control of cell type in yeast. Adv. Genet. 27, 33–62. 4043. Sprang, S.R. (1997). G protein mechanisms: insights from structural analysis. Annu. Rev. Biochem. 66, 639–678. 4044. Sprang, S.R. (1997). GAP into the breach. Science 277, 329– 330. ¨ 4045. Sprenger, F. and Nusslein-Volhard, C. (1992). Torso receptor activity is regulated by a diffusible ligand produced at the extracellular terminal regions of the Drosophila egg. Cell 71, 987–1001. ¨ 4046. Sprenger, F., Stevens, L.M., and Nusslein-Volhard, C. (1989). The Drosophila gene torso encodes a putative receptor tyrosine kinase. Nature 338, 478–483. 4047. Sprenger, F., Yakubovich, N., and O’Farrell, P.H. (1997).

REFERENCES

4048. 4049.

4050.

4051.

4052.

4053.

4054.

4055.

4056.

4057.

4058.

4059.

4060.

4061.

4062.

4063.

4064.

S-phase function of Drosophila cyclin A and its downregulation in G1 phase. Curr. Biol. 7, 488–499. Sprey, T.E. (1977). Aldehyde oxidase distribution in the imaginal discs of some diptera. W. Roux’s Arch. 183, 1–15. Sprey, T.E., Eskens, A.A.C., and Kuhn, D.T. (1982). Enzyme distribution patterns in the imaginal wing disc of Drosophila melanogaster and other diptera: A subdivision of compartments into territories. W. Roux’s Arch. 191, 301– 308. Sprey, T.E. and Kuhn, D.T. (1987). The regulation of aldehyde oxidase in imaginal wing discs of Drosophila hybrids: Evidence for cis- and trans-acting control elements. Genetics 115, 283–294. Sprey, T.E., Segal, D., Sprey-Pieters, H.E., and Kuhn, D.T. (1981). Inﬂuence of homoeotic genes on the aldehyde oxidase pattern in imaginal discs of Drosophila melanogaster. Dev. Genet. 2, 75–87. Spudich, J.L., Yang, C.-S., Jung, K.-H., and Spudich, E.N. (2000). Retinylidene proteins: structures and functions from archaea to humans. Annu. Rev. Cell Dev. Biol. 16, 365–392. Srinivasan, S., Peng, C.-Y., Nair, S., Skeath, J.B., Spana, E.P., and Doe, C.Q. (1998). Biochemical analysis of Prospero protein during asymmetric cell division: cortical Prospero is highly phosphorylated relative to nuclear Prospero. Dev. Biol. 204, 478–487. Srivastava, A., Mackay, J.O., and Bell, J.B. (2001). Rescue of a scalloped mutation using a Vestigial, Scalloped TEA domain chimera. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a203. ¨ St. Johnston, D. and Nusslein-Volhard, C. (1992). The origin of pattern and polarity in the Drosophila embryo. Cell 68, 201–219. St. Johnston, R.D., Hoffmann, F.M., Blackman, R.K., Segal, D., Grimaila, R., Padgett, R.W., Irick, H.A., and Gelbart, W.M. (1990). Molecular organization of the decapentaplegic gene in Drosophila melanogaster. Genes Dev. 4, 1114–1127. Stade, K., Ford, C.S., Guthrie, C., and Weis, K. (1997). Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90, 1041–1050. Staehling-Hampton, K., Hoffmann, F.M., Baylies, M.K., Rushton, E., and Bate, M. (1994). dpp induces mesodermal gene expression in Drosophila. Nature 372, 783–786. Staehling-Hampton, K., Laughon, A.S., and Hoffmann, F.M. (1995). A Drosophila protein related to the human zinc ﬁnger transcription factor PRDII/MBPI/HIV-EP1 is required for dpp signaling. Development 121, 3393–3403. ¨ Stahl, B., Diehlmann, A., and Sudhof, T.C. (1999). Direct interaction of Alzheimer’s disease-related Presenilin 1 with Armadillo protein p0071. J. Biol. Chem. 274 #14, 9141– 9148. Staley, J.P. and Guthrie, C. (1998). Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell 92, 315–326. Stambolic, V., Ruel, L., and Woodgett, J.R. (1996). Lithium inhibits glycogen synthase kinase-3 activity and mimics Wingless signalling in intact cells. Curr. Biol. 6, 1664–1668. Standley, H.J., Zorn, A.M., and Gurdon, J.B. (2001). eFGF and its mode of action in the community effect during Xenopus myogenesis. Development 128, 1347–1357. Stanford, N.P., Szczelkun, M.D., Marko, J.F., and Halford, S.E. (2000). One- and three-dimensional pathways for proteins to reach speciﬁc DNA sites. EMBO J. 19, 6546–6557.

417

4065. Stark, J., Bonacum, J., Remsen, J., and DeSalle, R. (1999). The evolution and development of dipteran wing veins: a systematic approach. Annu. Rev. Entomol. 44, 97–129. 4066. Stassen, M.J., Bailey, D., Nelson, S., Chinwalla, V., and Harte, P.J. (1995). The Drosophila trithorax proteins contain a novel variant of the nuclear receptor type DNA binding domain and an ancient conserved motif found in other chromosomal proteins. Mechs. Dev. 52, 209–223. 4067. Stauber, M., Prell, A., and Schmidt-Ott, U. (2001). Evolution of early developmental genes in the insect order Diptera. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a12. [Cf. 2002 Proc. Natl. Acad. Sci. USA 99, 274–279.] 4068. Stegman, M.A., Vallance, J.E., Elangovan, G., Sosinski, J., Cheng, Y., and Robbins, D.J. (2000). Identiﬁcation of a tetrameric Hedgehog signaling complex. J. Biol. Chem. 275 #29, 21809–21812. 4069. Steinberg, J. and Haglund, K. (1994). A screen door to understanding Drosophila genetics. (Landmark Interviews Series). J. NIH Res. 6, 68–77. 4070. Steinberg, M.S. (1963). Reconstruction of tissues by dissociated cells. Science 141, 401–408. 4071. Steinberg, M.S. (1970). Does differential adhesion govern self-assembly processes in histogenesis? Equilibrium conﬁgurations and the emergence of a hierarchy among populations of embryonic cells. J. Exp. Zool. 173, 395–434. 4072. Steinberg, M.S. (1981). The adhesive speciﬁcation of tissue self-organization. In “Morphogenesis and Pattern Formation,” (T.G. Connelly, L.L. Brinkley, and B.M. Carlson, eds.). Raven: New York, pp. 179–203. 4073. Steinberg, M.S. and Poole, T.J. (1981). Strategies for specifying form and pattern: adhesion-guided multicellular assembly. Phil. Trans. Roy. Soc. Lond. B 295, 451–460. 4074. Steinberg, M.S. and Poole, T.J. (1982). Cellular adhesive differentials as determinants of morphogenetic movements and organ segregation. In “Developmental Order: Its Origin and Regulation,” Symp. Soc. Dev. Biol., Vol. 40 (S. Subtelny and P.B. Green, eds.). Alan R. Liss: New York, pp. 351–378. 4075. Steindler, D.A. (1993). Glial boundaries in the developing nervous system. Annu. Rev. Neurosci. 16, 445–470. 4076. Steiner, E. (1976). Establishment of compartments in the developing leg imaginal discs of Drosophila melanogaster. W. Roux’s Arch. 180, 9–30. 4077. Steinmann-Zwicky, M. (1993). Sex determination in Drosophila: sis-b, a major numerator element of the X:A ratio in the soma, does not contribute to the X:A ratio in the germ line. Development 117, 763–767. 4078. Steller, H. (1995). Mechanisms and genes of cellular suicide. Science 267, 1445–1449. 4079. Steller, H., Fischbach, K.-F., and Rubin, G.M. (1987). disconnected: a locus required for neuronal pathway formation in the visual system of Drosophila. Cell 50, 1139–1153. 4080. Steller, H. and Grether, M.E. (1994). Programmed cell death in Drosophila. Neuron 13, 1269–1274. 4081. Stemerdink, C. and Jacobs, J.R. (1997). Argos and Spitz group genes function to regulate midline glial cell number in Drosophila embryos. Development 124, 3787–3796. 4082. Steneberg, P., Hemph¨al¨a, J., and Samakovlis, C. (1999). Dpp and Notch specify the fusion cell fate in dorsal branches of the Drosophila trachea. Mechs. Dev. 87, 153– 163. 4083. Stent, G.S. (1971). “Molecular Genetics: An Introductory Narrative.” W. H. Freeman, San Francisco. 4084. Stent, G.S. (1977). Explicit and implicit semantic content

418

4085.

4086. 4087. 4088. 4089. 4090. 4091.

4092.

4093. 4094. 4095. 4096. 4097.

4098.

4099. 4100. 4101. 4102. 4103.

4104.

4105.

4106.

4107.

4108.

4109.

REFERENCES

of the genetic information. In “Foundational Problems in the Special Sciences,” (R. Butts and J. Hintikka, eds.). D. Reidel: Dordrecht, Holland, pp. 131–149. Stent, G.S. (1981). Strength and weakness of the genetic approach to the development of the nervous system. Annu. Rev. Neurosci. 4, 163–194. Stent, G.S. (1985). The role of cell lineage in development. Phil. Trans. Roy. Soc. Lond. B 312, 3–19. Stent, G.S. (1998). Developmental cell lineage. Int. J. Dev. Biol. 42, 237–241. Stern, C. (1936). Genetics and ontogeny. Am. Nat. 70, 29– 35. Stern, C. (1936). Somatic crossing over and segregation in Drosophila melanogaster. Genetics 21, 625–730. Stern, C. (1940). The prospective signiﬁcance of imaginal discs in Drosophila. J. Morphol. 67, 107–122. Stern, C. (1940). Recent work on the relation between genes and developmental processes. Growth Suppl. 1, 19– 36. Stern, C. (1943). Genic action as studied by means of the effects of different doses and combinations of alleles. Genetics 28, 441–475. Stern, C. (1944). The journey, not the goal. Scientiﬁc Monthly 58, 96–100. Stern, C. (1948). The effects of changes in quantity, combination, and position of genes. Science 108, 615–621. Stern, C. (1954). Genes and developmental patterns. Proc. 9th Internat. Congr. Genetics Part I, 355–369. Stern, C. (1954). Two or three bristles. Am. Sci. 42, 213–247. Stern, C. (1956). The genetic control of developmental competence and morphogenetic tissue interactions in genetic mosaics. Roux’s Arch. Dev. Biol. 149, 1–25. Stern, C. (1956). Genetic mechanisms in the localized initiation of differentiation. Cold Spr. Harb. Symp. Quant. Biol. 21, 375–382. Stern, C. (1963). The cell lineage of the sternopleura in Drosophila melanogaster. Dev. Biol. 7, 365–378. Stern, C. (1968). “Genetic Mosaics and Other Essays.” Harvard Univ. Pr., Cambridge, Mass. Stern, C. (1969). Gene expression in genetic mosaics. Genetics 61 (Suppl.), 199–211. Stern, C. (1969). Richard Benedict Goldschmidt: April 12, 1878–April 24, 1958. Persp. Biol. Med. 12, 178–203. Stern, C. (1971). From crossing-over to developmental genetics. In “Stadler Genetics Symposia,” Vol. Vols. 1-2 (G. Kimbar and G. Redei, eds.). Univ. of Missouri Pr.: Columbia, pp. 21–28. Stern, C. (1974). A geneticist’s journey. In “Chromosomes and Cancer,” (J. German, ed.). Wiley & Sons: New York, pp. xii–xxv. Stern, C. and Hannah, A.M. (1950). The sex combs in gynanders of Drosophila melanogaster. Portug. Acta. Biol., Ser. A R. B. Goldschmidt Vol., 798–812. Stern, C. and Schaeffer, E.W. (1943). On primary attributes of alleles in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 29, 351–361. Stern, C. and Schaeffer, E.W. (1943). On wild-type isoalleles in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 29, 361–367. Stern, C. and Swanson, D.L. (1957). The control of the ocellar bristle by the scute locus in Drosophila melanogaster. J. Faculty Sci. (Zool.), Hokkaido Univ. Ser. 6, Vol. 13, 303– 307. Stern, C. and Tokunaga, C. (1967). Nonautonomy in differ-

4110. 4111. 4112.

4113. 4114.

4115. 4116. 4117. 4118.

4119.

4120.

4121.

4122.

4123.

4124. 4125.

4126.

4127.

4128.

4129.

4130.

entiation of pattern-determining genes in Drosophila. I. The sex comb of eyeless-Dominant. Proc. Natl. Acad. Sci. USA 57, 658–664. Stern, C. and Tokunaga, C. (1968). Autonomous pleiotropy in Drosophila. Proc. Natl. Acad. Sci. USA 60, 1252–1259. Stern, C.D. and Vasiliauskas, D. (1998). Clocked gene expression in somite formation. BioEssays 20, 528–531. Stern, D.L. (1998). A role of Ultrabithorax in morphological differences between Drosophila species. Nature 396, 463– 466. Stern, D.L. (2000). Evolutionary developmental biology and the problem of variation. Evolution 54, 1079–1091. Stern, D.L. and Emlen, D.J. (1999). The developmental basis for allometry in insects. Development 126, 1091– 1101. Sternberg, P.W. (1993). Falling off the knife edge. Curr. Biol. 3, 763–765. Sternberg, P.W. and Alberola-Ila, J. (1998). Conspiracy theory: RAS and RAF do not act alone. Cell 95, 447–450. Stevens, L. (1998). Twin peaks: Spitz and Argos star in patterning of the Drosophila egg. Cell 95, 291–294. Stevens, M.E. and Brower, D.L. (1986). Disruption of positional ﬁelds in apterous imaginal discs of Drosophila. Dev. Biol. 117, 326–330. Stevens, M.E. and Bryant, P.J. (1986). Temperaturedependent expression of the apterous phenotype in Drosophila melanogaster. Genetics 112, 217–228. Stevenson, R.D., Hill, M.F., and Bryant, P.J. (1995). Organ and cell allometry in Hawaiian Drosophila: how to make a big ﬂy. Proc. Roy. Soc. Lond. B 259, 105–110. Stewart, M., Murphy, C., and Fristrom, J.W. (1972). The recovery and preliminary characterization of X chromosome mutants affecting imaginal discs of Drosophila melanogaster. Dev. Biol. 27, 71–83. Stewart, S., Sundaram, M., Zhang, Y., Lee, J., Han, M., and Guan, K.-L. (1999). Kinase suppressor of Ras forms a multiprotein signaling complex and modulates MEK localization. Mol. Cell. Biol. 19, 5523–5534. Stochaj, U. and Rother, K.L. (1999). Nucleocytoplasmic trafﬁcking of proteins: with or without Ran? BioEssays 21, 579–589. Stocker, H. and Hafen, E. (2000). Genetic control of cell size. Curr. Opin. Gen. Dev. 10, 529–535. Stocker, R.F. (1994). The organization of the chemosensory system in Drosophila melanogaster : a review. Cell Tiss. Res. 275, 3–26. Stocker, R.F. and Gendre, N. (1988). Peripheral and central nervous effects of Lozenge 3 : a Drosophila mutant lacking basiconic antennal sensilla. Dev. Biol. 127, 12–24. Stocker, R.F., Gendre, N., and Batterham, P. (1993). Analysis of the antennal phenotype in the Drosophila mutant Lozenge. J. Neurogen. 9, 29–53. Stokoe, D., Macdonald, S.G., Cadwallader, K., Symons, M., and Hancock, J.F. (1994). Activation of Raf as a result of recruitment to the plasma membrane. Science 264, 1463– 1467. Stollewerk, A. (2000). Changes in cell shape in the ventral neuroectoderm of Drosophila melanogaster depend on the activity of the achaete-scute complex genes. Dev. Genes Evol. 210, 190–199. Stone, D.M., Hynes, M., Armanini, M., Swanson, T.A., Gu, Q., Johnson, R.L., Scott, M.P., Pennica, D., Goddard, A., Phillips, H., Noll, M., Hooper, J.E., de Sauvage, F., and Rosenthal, A. (1996). The tumor-suppressor gene patched

REFERENCES

4131.

4132.

4133.

4134.

4135.

4136.

4137.

4138. 4139.

4140.

4141.

4142.

4143.

4144. 4145. 4146.

4147.

4148. 4149.

4150.

encodes a candidate receptor for Sonic hedgehog. Nature 384, 129–134. Stowers, R.S., Russell, S., and Garza, D. (1999). The 82F late puff contains the L82 gene, an essential member of a novel gene family. Dev. Biol. 213, 116–130. Stowers, R.S. and Schwarz, T.L. (1999). A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype. Genetics 152, 1631– 1639. Strand, D., Jakobs, R., Merdes, G., Neumann, B., Kalmes, A., Heid, H.W., Husmann, I., and Mechler, B.M. (1994). The Drosophila lethal(2)giant larvae tumor suppressor protein forms homo-oligomers and is associated with nonmuscle myosin II heavy chain. J. Cell Biol. 127, 1361–1373. Strand, D., Raska, I., and Mechler, B.M. (1994). The Drosophila lethal(2)giant larvae tumor suppressor protein is a component of the cytoskeleton. J. Cell Biol. 127, 1345–1360. Streit, A. and Stern, C.D. (1999). Establishment and maintenance of the border of the neural plate in the chick: involvement of FGF and BMP activity. Mechs. Dev. 82, 51–66. Strigini, M. and Cohen, S.M. (1997). A Hedgehog activity gradient contributes to AP axial patterning of the Drosophila wing. Development 124, 4697–4705. Strigini, M. and Cohen, S.M. (1999). Formation of morphogen gradients in the Drosophila wing. Sems. Cell Dev. Biol. 10, 335–344. Strigini, M. and Cohen, S.M. (2000). Wingless gradient formation in the Drosophila wing. Curr. Biol. 10, 293–300. Str¨odicke, M., Karberg, S., and Korge, G. (2000). Domina (Dom), a new Drosophila member of the FKH/WH gene family, affects morphogenesis and is a suppressor of position-effect variegation. Mechs. Dev. 96, 67–78. Strub, S. (1977). Developmental potentials of the cells of the male foreleg disc of Drosophila. I. Pattern regulation in intact fragments. W. Roux’s Arch. 181, 309–320. Strub, S. (1977). Developmental potentials of the cells of the male foreleg disc of Drosophila. II. Regulative behaviour of dissociated fragments. W. Roux’s Arch. 182, 75–92. Strub, S. (1979). Heteromorphic regeneration in the developing imaginal primordia of Drosophila. In “Cell Lineage, Stem Cells and Cell Determination,” (N. Le Douarin, ed.). North-Holland Pub. Co.: Amsterdam, pp. 311–324. Strub, S. (1979). Leg regeneration in insects: an experimental analysis in Drosophila and a new interpretation. Dev. Biol. 69, 31–45. Struhl, G. (1977). Developmental compartments in the proboscis of Drosophila. Nature 270, 723–725. Struhl, G. (1981). Anterior and posterior compartments in the proboscis of Drosophila. Dev. Biol. 84, 372–385. Struhl, G. (1981). A blastoderm fate map of compartments and segments of the Drosophila head. Dev. Biol. 84, 386– 396. Struhl, G. (1981). A gene product required for correct initiation of segmental determination in Drosophila. Nature 293, 36–41. Struhl, G. (1981). A homoeotic mutation transforming leg to antenna in Drosophila. Nature 292, 635–638. Struhl, G. (1982). Genes controlling segmental speciﬁcation in the Drosophila thorax. Proc. Natl. Acad. Sci. USA 79, 7380–7384. Struhl, G. (1982). Spineless-aristapedia: a homeotic gene

419

4151.

4152. 4153. 4154.

4155. 4156.

4157.

4158.

4159. 4160.

4161.

4162.

4163.

4164. 4165. 4166.

4167.

4168.

4169.

4170.

4171.

that does not control the development of speciﬁc compartments in Drosophila. Genetics 102, 737–749. Struhl, G. (1983). Role of the esc+ gene product in ensuring the selective expression of segment-speciﬁc homeotic genes in Drosophila. J. Embryol. Exp. Morph. 76, 297–331. Struhl, G. (1984). Splitting the bithorax complex of Drosophila. Nature 308, 454–457. Struhl, G. (1984). A universal genetic key to body plan? Nature 310, 10–11. Struhl, G. (1989). Differing strategies for organizing anterior and posterior body pattern in Drosophila embryos. Nature 338, 741–744. Struhl, G. and Adachi, A. (1998). Nuclear access and action of Notch in vivo. Cell 93, 649–660. Struhl, G. and Adachi, A. (2000). Requirements for Presenilin-dependent cleavage of Notch and other transmembrane proteins. Molec. Cell 6, 625–636. [See also 2002 Dev. Cell 2, 69–78 and 79–89.] Struhl, G., Barbash, D.A., and Lawrence, P.A. (1997). Hedgehog acts by distinct gradient and signal relay mechanisms to organise cell type and cell polarity in the Drosophila abdomen. Development 124, 2155–2165. Struhl, G., Barbash, D.A., and Lawrence, P.A. (1997). Hedgehog organises the pattern and polarity of epidermal cells in the Drosophila abdomen. Development 124, 2143–2154. Struhl, G. and Basler, K. (1993). Organizing activity of wingless protein in Drosophila. Cell 72, 527–540. Struhl, G. and Brower, D. (1982). Early role of the esc+ gene product in the determination of segments in Drosophila. Cell 31, 285–292. Struhl, G., Fitzgerald, K., and Greenwald, I. (1993). Intrinsic activity of the Lin-12 and Notch intracellular domains in vivo. Cell 74, 331–345. Struhl, G. and Greenwald, I. (1999). Presenilin is required for activity and nuclear access of Notch in Drosophila. Nature 398, 522–525. [See also 2001 Proc. Natl. Acad. Sci. USA 98, 229–234.] Struhl, G., Struhl, K., and Macdonald, P.M. (1989). The gradient morphogen bicoid is a concentration-dependent transcriptional activator. Cell 57, 1259–1273. Struhl, K. (1991). Mechanisms for diversity in gene expression patterns. Neuron 7, 177–181. Struhl, K. (1999). Fundamentally different logic of gene regulation in eukaryotes and prokaryotes. Cell 98, 1–4. Strutt, D.I. (2001). Asymmetric localization of Frizzled and the establishment of cell polarity in the Drosophila wing. Molec. Cell 7, 367–375. Strutt, D.I. and Mlodzik, M. (1995). Ommatidial polarity in the Drosophila eye is determined by the direction of furrow progression and local interactions. Development 121, 4247–4256. Strutt, D.I. and Mlodzik, M. (1996). The regulation of hedgehog and decapentaplegic during Drosophila eye imaginal disc development. Mechs. Dev. 58, 39–50. Strutt, D.I. and Mlodzik, M. (1997). Hedgehog is an indirect regulator of morphogenetic furrow progression in the Drosophila eye disc. Development 124, 3233–3240. Strutt, D.I., Weber, U., and Mlodzik, M. (1997). The role of RhoA in tissue polarity and Frizzled signalling. Nature 387, 292–295. Strutt, D.I., Wiersdorff, V., and Mlodzik, M. (1995). Regulation of furrow progression in the Drosophila eye by cAMP-dependent protein kinase A. Nature 373, 705–709.

420

4172. Strutt, H., Cavalli, G., and Paro, R. (1997). Co-localization of Polycomb protein and GAGA factor on regulatory elements responsible for the maintenance of homeotic gene expression. EMBO J. 16, 3621–3632. 4173. Strutt, H. and Strutt, D. (1999). Polarity determination in the Drosophila eye. Curr. Opin. Gen. Dev. 9, 442–446. 4174. Strutt, H., Thomas, C., Nakano, Y., Stark, D., Neave, B., Taylor, A.M., and Ingham, P.W. (2001). Mutations in the sterol-sensing domain of Patched suggest a role for vesicular trafﬁcking in Smoothened regulation. Curr. Biol. 11, 608–613. 4175. Stuart, E.T., Kioussi, C., and Gruss, P. (1993). Mammalian Pax genes. Annu. Rev. Genet. 27, 219–236. 4176. Stukenberg, P.T., Lustig, K.D., McGarry, T.J., King, R.W., Kuang, J., and Kirschner, M.W. (1997). Systematic identiﬁcation of mitotic phosphoproteins. Curr. Biol. 7, 338–348. 4177. Sturm, R.A. and Herr, W. (1988). The POU domain is a bipartite DNA-binding structure. Nature 336, 601–604. 4178. Sturtevant, A.H. (1918). An analysis of the effects of selection. Carnegie Inst. Wash. Publ. 264, 1–68. 4179. Sturtevant, A.H. (1925). The effects of unequal crossingover at the Bar locus in Drosophila. Genetics 10, 117–147. 4180. Sturtevant, A.H. (1929). The claret mutant type of Drosophila simulans: a study of chromosome elimination and of cell-lineage. Z. wiss. Zool. 135, 323–356. 4181. Sturtevant, A.H. (1932). The use of mosaics in the study of the developmental effects of genes. Proc. 6th Internat. Congr. Genet. 1, 304–307. 4182. Sturtevant, A.H. (1961). “Genetics and Evolution. Selected papers of A. H. Sturtevant.” W. H. Freeman, San Francisco. (N. B.: Sturtevant’s retort to Stern’s argument is on p. 145.) 4183. Sturtevant, A.H. (1965). The “ﬂy room.” Am. Sci. 53, 303– 307. [See also 2001 Genetics 159, 1–5.] 4184. Sturtevant, A.H. (1965). “A History of Genetics.” Harper & Row, New York. 4185. Sturtevant, A.H. (1970). Studies on the bristle pattern of Drosophila. Dev. Biol. 21, 48–61. 4186. Sturtevant, A.H. and Morgan, T.H. (1923). Reverse mutation of the Bar gene correlated with crossing over. Science 57, 746–747. 4187. Sturtevant, A.H. and Schultz, J. (1931). The inadequacy of the sub-gene hypothesis of the nature of the scute allelomorphs of Drosophila. Proc. Natl. Acad. Sci. U. S. 17, 265–270. 4188. Sturtevant, M.A., Biehs, B., Marin, E., and Bier, E. (1997). The spalt gene links the A/P compartment boundary to a linear adult structure in the Drosophila wing. Development 124, 21–32. 4189. Sturtevant, M.A. and Bier, E. (1995). Analysis of the genetic hierarchy guiding wing vein development in Drosophila. Development 121, 785–801. 4190. Sturtevant, M.A., O’Neill, J.W., and Bier, E. (1994). Downregulation of Drosophila Egf-r mRNA levels following hyperactivated receptor signaling. Development 120, 2593– 2600. 4191. Sturtevant, M.A., Roark, M., and Bier, E. (1993). The Drosophila rhomboid gene mediates the localized formation of wing veins and interacts genetically with components of the EGF-R signaling pathway. Genes Dev. 7, 961– 973. 4192. Sturtevant, M.A., Roark, M., O’Neill, J.W., Biehs, B., Colley, N., and Bier, E. (1996). The Drosophila Rhomboid protein is concentrated in patches at the apical cell surface. Dev. Biol. 174, 298–309. ¨ 4193. Stuttem, I. and Campos-Ortega, J.A. (1997). Autonomous

REFERENCES

4194.

4195.

4196. 4197.

4198.

4199.

4200. 4201.

4202.

4203.

4204. 4205.

4206.

4207.

4208.

4209.

4210.

4211.

and non-autonomous phenotypic expression of Notch mutant cells in Drosophila embryogenesis. Dev. Genes Evol. 207, 82–89. Su, M.-T., Fujioka, M., Goto, T., and Bodmer, R. (1999). The Drosophila homeobox genes zfh-1 and even-skipped are required for cardiac-speciﬁc differentiation of a numbdependent lineage decision. Development 126, 3241– 3251. Su, M.A., Wisotzkey, R.G., and Newfeld, S.J. (2001). A screen for modiﬁers of decapentaplegic mutant phenotypes identiﬁes lilliputian, the only member of the FragileX/Burkitt’s Lymphoma family of transcription factors in Drosophila melanogaster. Genetics 157, 717–725. Su, T.T. and O’Farrell, P.H. (1998). Size control: cell proliferation does not equal growth. Curr. Biol. 8, R687–R689. Subramaniam, V., Bomze, H.M., and L´opez, A.J. (1994). Functional differences between Ultrabithorax protein isoforms in Drosophila melanogaster: evidence from elimination, substitution and ectopic expression of speciﬁc isoforms. Genetics 136, 979–991. Subramaniam, V., Jovin, T.M., and Rivera-Pomar, R.V. (2001). Aromatic amino acids are critical for stability of the Bicoid homeodomain. J. Biol. Chem. 276 #24, 21506– 21511. Sudarsanam, P. and Winston, F. (2000). The Swi/Snf family: nucleosome-remodeling complexes and transcriptional control. Trends Genet. 16, 345–351. Sudol, M. and Hunter, T. (2000). New wrinkles for an old domain. Cell 103, 1001–1004. Sulston, J.E., Albertson, D.G., and Thomson, J.N. (1980). The Caenorhabditis elegans male: postembryonic development of nongonadal structures. Dev. Biol. 78, 542–576. Sulston, J.E., Schierenberg, E., White, J.G., and Thomson, J.N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64–119. Sun, B., Hursh, D.A., Jackson, D., and Beachy, P.A. (1995). Ultrabithorax protein is necessary but not sufﬁcient for full activation of decapentaplegic expression in the visceral mesoderm. EMBO J. 14, 520–535. Sun, F.-L. and Elgin, S.C.R. (1999). Putting boundaries on silence. Cell 99, 459–462. Sun, H., Charles, C.H., Lau, L.F., and Tonks, N.K. (1993). MKP-1 (3CH134), an immediate early gene product, is a dual speciﬁcity phosphatase that dephosphorylates MAP kinase in vivo. Cell 75, 487–493. Sun, X. and Artavanis-Tsakonas, S. (1996). The intracellular deletions of DELTA and SERRATE deﬁne dominant negative forms of the Drosophila Notch ligands. Development 122, 2465–2474. Sun, X. and Artavanis-Tsakonas, S. (1997). Secreted forms of DELTA and SERRATE deﬁne antagonists of Notch signaling in Drosophila. Development 124, 3439–3448. Sun, Y., Jan, L.Y., and Jan, Y.N. (1998). Transcriptional regulation of atonal during development of the Drosophila peripheral nervous system. Development 125, 3731– 3740. Sun, Y., Jan, L.Y., and Jan, Y.N. (2000). Ectopic scute induces Drosophila ommatidia development without R8 founder photoreceptors. Proc. Natl. Acad. Sci. USA 97, 6815–6819. Sun, Y.H., Tsai, C.-J., Green, M.M., Chao, J.-L., Yu, C.-T., Jaw, T.J., Yeh, J.-Y., and Bolshakov, V.N. (1995). white as a reporter gene to detect transcriptional silencers specifying position-speciﬁc gene expression during Drosophila melanogaster eye development. Genetics 141, 1075–1086. Sunio, A., Metcalf, A.B., and Kr¨amer, H. (1999). Genetic

REFERENCES

4212.

4213.

4214. 4215.

4216.

4217.

4218.

4219.

4220.

4221.

4222.

4223.

4224.

4225. 4226.

4227.

4228.

dissection of endocytic trafﬁcking in Drosophila using a horseradish peroxidase-Bride of sevenless chimera: hook is required for normal maturation of multivesicular endosomes. Mol. Biol. Cell 10, 847–859. [Cf. 2002 Cell 108, 261–269.] Sunkel, C.E. and Whittle, J.R.S. (1987). Brista: a gene involved in the speciﬁcation and differentiation of distal cephalic and thoracic structures in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 196, 124–132. Sutrias-Grau, M. and Arnosti, D.N. (2001). Functional characterization of the dCtBP corepressor in the Drosophila embryo. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a126. [Cf. 2001 Dev. Biol. 239, 229–240.] Suzuki, D.T. (1970). Temperature-sensitive mutations in Drosophila melanogaster. Science 170, 695–706. Suzuki, T., Maurel-Zaffran, C., Gahmon, G., and Dickson, B.J. (2001). The receptor tyrosine phosphatase DLAR controls R7 axon targeting in the Drosophila visual system. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a253. [Cf. 2001 Neuron 32, 225–235.] Suzuki, T. and Saigo, K. (2000). Transcriptional regulation of atonal required for Drosophila larval eye development by concerted action of Eyes absent, Sine oculis and Hedgehog signaling independent of Fused kinase and Cubitus interruptus. Development 127, 1531–1540. Svendsen, P.C., Marshall, S.D.G., Kyba, M., and Brook, W.J. (2000). The combgap locus encodes a zinc-ﬁnger protein that regulates cubitus interruptus during limb development in Drosophila melanogaster. Development 127, 4083–4093. Swammerdam, J. (1758). “The Book of Nature.” (T. Flloyd, translator), C. G. Seyffert, London. (Reprint Ed., 1978, Arno Press, N. Y.). Swindells, M.B., Orengo, C.A., Jones, D.T., Hutchinson, E.G., and Thornton, J.M. (1998). Contemporary approaches to protein structure classiﬁcation. BioEssays 20, 884–891. Symes, K., Yord´an, C., and Mercola, M. (1994). Morphological differences in Xenopus embryonic mesodermal cells are speciﬁed as an early response to distinct threshold concentrations of activin. Development 120, 2339–2346. Szabad, J. and Bryant, P.J. (1982). The mode of action of “discless” mutations in Drosophila melanogaster. Dev. Biol. 93, 240–256. Szabad, J., Schpbach, T., and Wieschaus, E. (1979). Cell lineage and development in the larval epidermis of Drosophila melanogaster. Dev. Biol. 73, 256–271. Szabad, J., Simpson, P., and N¨othiger, R. (1979). Regeneration and compartments in Drosophila. J. Embryol. Exp. Morph. 49, 229–241. Szab´o, K., J´ekely, G., and Rørth, P. (2001). Cloning and expression of sprint, a Drosophila homologue of RIN1. Mechs. Dev. 101, 259–262. Szebenyi, A.L. (1969). Cleaning behaviour in Drosophila melanogaster. Anim. Behav. 17, 641–651. ¨ D., Eresh, S., and Bienz, M. (1998). Functional inSzuts, tertwining of Dpp and EGFR signaling during Drosophila endoderm induction. Genes Dev. 12, 2022–2035. Tabata, T., Eaton, S., and Kornberg, T.B. (1992). The Drosophila hedgehog gene is expressed speciﬁcally in posterior compartment cells and is a target of engrailed regulation. Genes Dev. 6, 2635–2645. Tabata, T. and Kornberg, T.B. (1994). Hedgehog is a signaling protein with a key role in patterning Drosophila imaginal discs. Cell 76, 89–102.

421

4229. Tabata, T., Schwartz, C., Gustavson, E., Ali, Z., and Kornberg, T.B. (1995). Creating a Drosophila wing de novo, the role of engrailed, and the compartment border hypothesis. Development 121, 3359–3369. 4230. Tabin, C.J., Carroll, S.B., and Panganiban, G. (1999). Out on a limb: parallels in vertebrate and invertebrate limb patterning and the origin of appendages. Amer. Zool. 39, 650–663. [See also 2001 Evol. Dev. 3, 343–354.] 4231. Taghert, P.H., Doe, C.Q., and Goodman, C.S. (1984). Cell determination and regulation during development of neuroblasts and neurones in grasshopper embryo. Nature 307, 163–165. 4232. Tago, K.-i., Nakamura, T., Nishita, M., Hyodo, J., Nagai, S.-i., Murata, Y., Adachi, S., Ohwada, S., Morishita, Y., Shibuya, H., and Akiyama, T. (2000). Inhibition of Wnt signaling by ICAT, a novel β-catenin-interacting protein. Genes Dev. 14, 1741–1749. 4233. Taguchi, A., Sawamoto, K., and Okano, H. (2000). Mutations modulating the argos-regulated signaling pathway in Drosophila eye development. Genetics 154, 1639– 1648. 4234. Tahirov, T.H., Inoue-Bungo, T., Morii, H., Fujikawa, A., Sasaki, M., Kimura, K., Shiina, M., Sato, K., Kumasaka, T., Yamamoto, M., Ishii, S., and Ogata, K. (2001). Structural analyses of DNA recognition by the AML1/Runx-1 Runt domain and its allosteric control by CBFβ. Cell 104, 755– 767. 4235. Takagaki, Y. and Manley, J.L. (1994). A polyadenlyation factor subunit is the human homologue of the Drosophila suppressor of forked protein. Nature 372, 471–474. 4236. Takahashi, K., Matsuo, T., Katsube, T., Ueda, R., and Yamamoto, D. (1998). Direct binding between two PDZ domain proteins Canoe and ZO-1 and their roles in regulation of the Jun N-terminal kinase pathway in Drosophila morphogenesis. Mechs. Dev. 78, 97–111. 4237. Takahisa, M., Togashi, S., Suzuki, T., Kobayashi, M., Murayama, A., Kondo, K., Miyake, T., and Ueda, R. (1996). The Drosophila tamou gene, a component of the activating pathway of extramacrochaetae expression, encodes a protein homologous to mammalian cell-cell junctionassociated protein ZO-1. Genes Dev. 10, 1783–1795. 4238. Takano, T.S. (1998). Loss of notum macrochaetae as an interspeciﬁc hybrid anomaly between Drosophila melanogaster and D. simulans. Genetics 149, 1435–1450. 4239. Takano-Shimizu, T. (2000). Genetic screens for factors involved in the notum bristle loss of interspeciﬁc hybrids between Drosophila melanogaster and D. simulans. Genetics 156, 269–282. 4240. Takei, K., McPherson, P.S., Schmid, S.L., and De Camilli, P. (1995). Tubular membrane invaginations coated by dynamin rings are induced by GTP-γS in nerve terminals. Nature 374, 186–190. 4241. Talbert, P.B. and Garber, R.L. (1994). The Drosophila homeotic mutation Nasobemia (Antp Ns ) and its revertants: an analysis of mutational reversion. Genetics 138, 709–720. 4242. Tamai, K., Semenov, M., Kato, Y., Spokony, R., Liu, C., Katsuyama, Y., Hess, F., Saint-Jeannet, J.-P., and He, X. (2000). LDL-receptor-related proteins in Wnt signal transduction. Nature 407, 530–535. 4243. Tamkun, J.W., Deuring, R., Scott, M.P., Kissinger, M., Pattatucci, A.M., Kaufman, T.C., and Kennison, J.A. (1992). brahma: a regulator of Drosophila homeotic genes structurally related to the yeast transcriptional activator SNF2/SWI2. Cell 68, 561–572.

422

4244. Tamura, K., Taniguchi, Y., Minoguchi, S., Sakai, T., Tun, T., Furukawa, T., and Honjo, T. (1995). Physical interaction between a novel domain of the receptor Notch and the transcription factor RBP-Jκ/Su(H). Curr. Biol. 5, 1416– 1423. 4245. Tan, P.B.O. and Kim, S.K. (1999). Signaling speciﬁcity: the RTK/RAS/MAP kinase pathway in metazoans. Trends Genet. 15, 145–149. 4246. Tanaka Hall, T.M., Porter, J.A., Young, K.E., Koonin, E.V., Beachy, P.A., and Leahy, D.J. (1997). Crystal structure of a Hedgehog autoprocessing domain: homology between Hedgehog and self-splicing proteins. Cell 91, 8597. 4247. Tanaka-Matakatsu, M. and Thomas, B. (2001). Shattered is a new rough eye mutant which interacts with roughex, string and ﬁzzy-related. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a66. 4248. Tanenbaum, S.B., Gorski, S.M., Rusconi, J.C., and Cagan, R.L. (2000). A screen for dominant modiﬁers of the irreCrst cell death phenotype in the developing Drosophila retina. Genetics 156, 205–217. 4249. Tang, A.H., Neufeld, T.P., Kwan, E., and Rubin, G.M. (1997). PHYL acts to down-regulate TTK88, a transcriptional repressor of neuronal cell fates, by a SINA-dependent mechanism. Cell 90, 459–467. ¨ 4250. Tang, A.H., Neufeld, T.P., Rubin, G.M., and Muller, H.-A.J. (2001). Transcriptional regulation of cytoskeletal functions and segmentation by a novel maternal pair-rule gene, lilliputian. Development 128, 801–813. 4251. Tanimoto, H., Itoh, S., ten Dijke, P., and Tabata, T. (2000). Hedgehog creates a gradient of DPP activity in Drosophila wing imaginal discs. Molec. Cell 5, 59–71. 4252. Tanoue, T., Adachi, M., Moriguchi, T., and Nishida, E. (2000). A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nature Cell Biol. 2, 110–116. 4253. Tanoue, T., Maeda, R., Adachi, M., and Nishida, E. (2001). Identiﬁcation of a docking groove on ERK and p38 MAP kinases that regulates the speciﬁcity of docking interactions. EMBO J. 20, 466–479. 4254. Tashiro, S., Michiue, T., Higashijima, S.-i., Zenno, S., Ishimaru, S., Takahashi, F., Orihara, M., Kojima, T., and Saigo, K. (1993). Structure and expression of hedgehog, a Drosophila segment-polarity gene required for cell-cell communication. Gene 124, 183–189. 4255. Tata, F. and Hartley, D.A. (1993). The role of the Enhancer of split complex during cell fate determination in Drosophila. Development 1993 Suppl., 139–148. 4256. Tata, F. and Hartley, D.A. (1995). Inhibition of cell fate in Drosophila by Enhancer of split genes. Mechs. Dev. 51, 305– 315. 4257. Taubert, H. and Szabad, J. (1987). Genetic control of cell proliferation in female germ line cells of Drosophila: Mosaic analysis of ﬁve discless mutations. Mol. Gen. Genet. 209, 545–551. 4258. Tautz, D. (1996). Selector genes, polymorphisms, and evolution. Science 271, 160–161. 4259. Tautz, D. (2000). Evolution of transcriptional regulation. Curr. Opin. Gen. Dev. 10, 575–579. [See also 2001 Evol. Dev. 3, 109–119.] 4260. Tautz, D. (2000). A genetic uncertainty problem. Trends Genet. 16, 475–477. 4261. Tayler, T.D. and Garrity, P.A. (2001). Regulation of R1-R6 photoreceptor axon targeting. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a253.

REFERENCES

4262. Taylor, A.M., Nakano, Y., Mohler, J., and Ingham, P.W. (1993). Contrasting distributions of patched and hedgehog proteins in the Drosophila embryo. Mechs. Dev. 42, 89–96. 4263. Taylor, J., Abramova, N., Charlton, J., and Adler, P.N. (1998). Van Gogh: a new Drosophila tissue polarity gene. Genetics 150, 199–210. 4264. Tear, G. (1999). Neuronal guidance: a genetic perspective. Trends Genet. 15, 113–118. 4265. Teleman, A.A. and Cohen, S.M. (2000). Dpp gradient formation in the Drosophila wing imaginal disc. Cell 103, 971–980. 4266. Telford, M.J. and Thomas, R.H. (1998). Of mites and zen: expression studies in a chelicerate arthropod conﬁrm zen is a divergent Hox gene. Dev. Genes Evol. 208, 591–594. 4267. ten Dijke, P., Miyazono, K., and Heldin, C.-H. (1996). Signalling via hetero-oligomeric complexes of type I and type II serine/threonine kinase receptors. Curr. Opin. Cell Biol. 8, 139–145. 4268. Tepass, U., Gruszynski-DeFeo, E., Haag, T.A., Omatyar, L., T¨or¨ok, T., and Hartenstein, V. (1996). shotgun encodes Drosophila E-cadherin and is preferentially required during cell rearrangement in the neurectoderm and other morphogenetically active epithelia. Genes Dev. 10, 672–685. 4269. Tepass, U. and Hartenstein, V. (1995). Neurogenic and proneural genes control cell fate speciﬁcation in the Drosophila endoderm. Development 121, 393–405. 4270. Tepass, U., Pellikka, M., Tanentzapf, G., Pinto, M., Hong, H., Smith, C., McGlade, J., and Ready, D. (2001). Crumbs, the Drosophila homolog of human CRB1 (retinitis pigmentosa 12/RP12), is required for photoreceptor cell morphogenesis. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a45. 4271. Terracol, R. and Lengyel, J.A. (1994). The thick veins gene of Drosophila is required for dorsoventral polarity of the embryo. Genetics 138, 165–178. 4272. Thackeray, J.R., Gaines, P.C.W., Ebert, P., and Carlson, J.R. (1998). small wing encodes a phospholipase C-γ that acts as a negative regulator of R7 development in Drosophila. Development 125, 5033–5042. 4273. Thaker, H.M. and Kankel, D.R. (1992). Mosaic analysis gives an estimate of the extent of genomic involvement in the development of the visual system in Drosophila melanogaster. Genetics 131, 883–894. 4274. Thanos, D. and Maniatis, T. (1995). Virus induction of human IFNβ gene expression requires the assembly of an enhanceosome. Cell 83, 1091–1100. 4275. The, I., Bellaiche, Y., and Perrimon, N. (1999). Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan. Molec. Cell 4, 633–639. 4276. The, I. and Perrimon, N. (2000). Morphogen diffusion: the case of the Wingless protein. Nature Cell Biol. 2, E79– E82. 4277. Theisen, H., Haerry, T.E., O’Connor, M.B., and Marsh, J.L. (1996). Developmental territories created by mutual antagonism between Wingless and Decapentaplegic. Development 122, 3939–3948. 4278. Theisen, H., Purcell, J., Bennett, M., Kansagara, D., Syed, A., and Marsh, J.L. (1994). dishevelled is required during wingless signaling to establish both cell polarity and cell identity. Development 120, 347–360. 4279. Theodosiou, N.A., Zhang, S., Wang, W.-Y., and Xu, T. (1998). slimb coordinates wg and dpp expression in the

REFERENCES

4280.

4281.

4282.

4283.

4284.

4285.

4286.

4287.

4288.

4289.

4290.

4291.

4292. 4293.

4294. 4295. 4296.

dorsal-ventral and anterior-posterior axes during limb development. Development 125, 3411–3416. Th´erond, P., Alves, G., Limbourg-Bouchon, B., Tricoire, H., Guillemet, E., Brissard-Zahraoui, J., Lamour-Isnard, C., and Busson, D. (1996). Functional domains of Fused, a serine-threonine kinase required for signaling in Drosophila. Genetics 142, 1181–1198. Therond, P., Busson, D., Guillemet, E., LimbourgBouchon, B., Preat, T., Terracol, R., Tricoire, H., and Lamour-Isnard, C. (1993). Molecular organisation and expression pattern of the segment polarity gene fused of Drosophila melanogaster. Mechs. Dev. 44, 65–80. Th´erond, P.P., Bouchon, B.L., Gallet, A., Dussilol, F., Pietri, T., van den Heuvel, M., and Tricoire, H. (1999). Differential requirements of the Fused kinase for Hedgehog signalling in the Drosophila embryo. Development 126, 4039– 4051. Th´erond, P.P., Knight, J.D., Kornberg, T.B., and Bishop, J.M. (1996). Phosphorylation of the fused protein kinase in response to signaling from hedgehog. Proc. Natl. Acad. Sci. USA 93, 4224–4228. Therrien, M., Chang, H.C., Solomon, N.M., Karim, F.D., Wassarman, D.A., and Rubin, G.M. (1995). KSR, a novel protein kinase required for RAS signal transduction. Cell 83, 879–888. Therrien, M., Michaud, N.R., Rubin, G.M., and Morrison, D.K. (1996). KSR modulates signal propagation within the MAPK cascade. Genes Dev. 10, 2684–2695. Therrien, M., Morrison, D.K., Wong, A.M., and Rubin, G.M. (2000). A genetic screen for modiﬁers of a kinase suppressor of Ras-dependent rough eye phenotype in Drosophila. Genetics 156, 1231–1242. Therrien, M., Wong, A.M., Kwan, E., and Rubin, G.M. (1999). Functional analysis of CNK in RAS signaling. Proc. Natl. Acad. Sci. USA 96, 13259–13263. Therrien, M., Wong, A.M., and Rubin, G.M. (1998). CNK, a RAF-binding multidomain protein required for RAS signaling. Cell 95, 343–353. Thieffry, D., Huerta, A.M., P´erez-Rueda, E., and ColladoVides, J. (1998). From speciﬁc gene regulation to genomic networks: a global analysis of transcriptional regulation in Escherichia coli. BioEssays 20, 433–440. Thisse, C., Perrin-Schmitt, F., Stoetzel, C., and Thisse, B. (1991). Sequence-speciﬁc transactivation of the Drosophila twist gene by the dorsal gene product. Cell 65, 1191–1201. Thomas, B.J., Gunning, D.A., Cho, J., and Zipursky, S.L. (1994). Cell cycle progression in the developing Drosophila eye: roughex encodes a novel protein required for the establishment of G1 . Cell 77, 1003–1014. Thomas, B.J. and Wassarman, D.A. (1999). A ﬂy’s eye view of biology. Trends Genet. 15, 184–190. Thomas, B.J., Zavitz, K.H., Dong, X., Lane, M.E., Weigmann, K., Finley, R.L., Jr., Brent, R., Lehner, C.F., and Zipursky, S.L. (1997). roughex down-regulates G2 cyclins in G1 . Genes Dev. 11, 1289–1298. Thomas, D. and Tyers, M. (2000). Transcriptional regulation: kamikazi activators. Curr. Biol. 10, R341–R343. Thomas, G.H. (2001). Spectrin: the ghost in the machine. BioEssays 23, 152–160. Thomas, G.H., Zarnescu, D.C., Juedes, A.E., Bales, M.A., Londergan, A., Korte, C.C., and Kiehart, D.P. (1998). Drosophila βHeavy -spectrin is essential for development and contributes to speciﬁc cell fates in the eye. Development 125, 2125–2134.

423

4297. Thomas, J.B., Crews, S.T., and Goodman, C.S. (1988). Molecular genetics of the single-minded locus: a gene involved in the development of the Drosophila nervous system. Cell 52, 133–141. 4298. Thomas, J.H. (1993). Thinking about genetic redundancy. Trends Genet. 9, 395–399. 4299. Thomas, J.O. and Travers, A.A. (2001). HMG1 and 2, and related “architectural” DNA-binding proteins. Trends Biochem. Sci. 26, 167–174. 4300. Thomas, U., Ebitsch, S., Gorczyca, M., Koh, Y.H., Hough, C.D., Woods, D., Gundelﬁnger, E.D., and Budnik, V. (2000). Synaptic targeting and localization of Discs-large is a stepwise process controlled by different domains of the protein. Curr. Biol. 10, 1108–1117. 4301. Thomas, U., J¨onsson, F., Speicher, S.A., and Knust, E. (1995). Phenotypic and molecular characterization of SerD, a dominant allele of the Drosophila gene Serrate. Genetics 139, 203–213. 4302. Thomas, U., Speicher, S.A., and Knust, E. (1991). The Drosophila gene Serrate encodes an EGF-like transmembrane protein with a complex expression pattern in embryos and wing discs. Development 111, 749–761. 4303. Thompson, A. and Kavaler, J. (2001). The role of Pax 2 in glial cell differentiation during Drosophila sense organ development. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a262. 4304. Thompson, C.C. and McKnight, S.L. (1992). Anatomy of an enhancer. Trends Genet. 8, 232–236. 4305. Thompson, D.W. (1913). “On Aristotle as a Biologist.” Clarendon Pr., Oxford. 4306. Thompson, D.W. (1917). “On Growth and Form.” Cambridge Univ. Pr., Cambridge. 4307. Thompson, J.N., Jr. (1974). Studies on the nature and function of polygenic loci in Drosophila. II. The subthreshold wing vein pattern revealed in selection experiments. Heredity 33, 389–401. 4308. Thompson, J.N., Jr., Hellack, J.J., and Kennedy, J.S. (1982). Polygenic analysis of pattern formation in Drosophila: speciﬁcation of campaniform sensilla positions on the wing. Dev. Genet. 3, 115–128. 4309. Thompson, J.N., Jr., Spivey, W.E., and Duncan, D.K. (1985). Temperature-dependent responses to a developmental gradient in the Drosophila wing. Experientia 41, 1346– 1347. 4310. Thor, S., Andersson, S.G.E., Tomlinson, A., and Thomas, J.B. (1999). A LIM-homeodomain combinatorial code for motor-neuron pathway selection. Nature 397, 76– 80. 4311. Thummel, C.S. (1996). Flies on steroids–Drosophila metamorphosis and the mechanisms of steriod hormone action. Trends Genet. 12, 306–310. [See also 2001 Dev. Cell. 1, 453–465.] ¨ 4312. Thuringer, F., Cohen, S.M., and Bienz, M. (1993). Dissection of an indirect autoregulatory response of a homeotic Drosophila gene. EMBO J. 12, 2419–2430. 4313. Tie, F., Furuyama, T., and Harte, P.J. (1998). The Drosophila Polycomb Group proteins ESC and E(Z) bind directly to each other and co-localize at multiple chromosome sites. Development 125, 3483–3496. 4314. Tie, F., Furuyama, T., Prasad-Sinha, J., Jane, E., and Harte, P. (2001). Puriﬁcation and characterization of components of the ESC complex. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a90. 4315. Tie, F., Furuyama, T., Prasad-Sinha, J., Jane, E., and Harte, P.J. (2001). The Drosophila Polycomb Group proteins ESC

424

4316.

4317.

4318.

4319.

4320.

4321.

4322.

4323.

4324.

4325.

4326.

4327.

4328.

4329.

4330.

4331.

REFERENCES

and E(Z) are present in a complex containing the histonebinding protein p55 and the histone deacetylase RPD3. Development 128, 275–286. [See also 2001 Proc. Natl. Acad. Sci. USA 98, 9730–9735.] Tie, F. and Harte, P. (2001). Characterization of ESC self-association and phosphorylation. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a98. Tien, A.-C., Hsei, H.-Y., and Chien, C.-T. (1999). Dynamic expression and cellular localization of the Drosophila 143-3 during embryonic development. Mechs. Dev. 81, 209– 212. Tietze, K., Oellers, N., and Knust, E. (1992). Enhancer of splitD , a dominant mutation of Drosophila, and its use in the study of functional domains of a helix-loop-helix protein. Proc. Natl. Acad. Sci. USA 89, 6152–6156. Tillib, S., Petruk, S., Sedkov, Y., Kuzin, A., Fujioka, M., Goto, T., and Mazo, A. (1999). Trithorax- and Polycomb-group response elements within an Ultrabithorax transcription maintenance unit consist of closely situated but separable sequences. Mol. Cell. Biol. 19, 5189–5202. Timms, J.F., Swanson, K.D., Marie-Cardine, A., Raab, M., Rudd, C.E., Schraven, B., and Neel, B.G. (1999). SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages. Curr. Biol. 9, 927–930. Tio, M., Ma, C., and Moses, K. (1994). spitz, a Drosophila homolog of transforming growth factor-α, is required in the founding photoreceptor cells of the compound eye facets. Mechs. Dev. 48, 13–23. Tio, M. and Moses, K. (1997). The Drosophila TGFα homolog Spitz acts in photoreceptor recruitment in the developing retina. Development 124, 343–351. Tio, M., Zavortink, M., Yang, X., and Chia, W. (1999). A functional analysis of inscuteable and its roles during Drosophila asymmetric cell divisions. J. Cell Sci. 112, 1541– 1551. Tiong, S.Y., Girton, J.R., Hayes, P.H., and Russell, M.A. (1977). Effect of regeneration on compartment speciﬁcity of the bithorax mutant of Drosophila melanogaster. Nature 268, 435–437. Tiong, S.Y.K., Nash, D., and Bender, W. (1995). Dorsal wing, a locus that affects dorsoventral wing patterning in Drosophila. Development 121, 1649–1656. [See also 2001 Development 128, 3263–3268.] Tiong, S.Y.K. and Russell, M.A. (1986). Effect of the bithorax mutation on determination in duplicating Drosophila imaginal discs. Dev. Biol. 113, 271–281. Tiong, S.Y.K. and Russell, M.A. (1990). Clonal analysis of segmental and compartmental homoeotic transformations in Polycomb mutants of Drosophila melanogaster. Dev. Biol. 141, 306–318. Tiong, S.Y.K., Whittle, J.R.S., and Gribbin, M.C. (1987). Chromosomal continuity in the abdominal region of the bithorax complex of Drosophila is not essential for its contribution to metameric identity. Development 101, 135– 142. Tissot, M. and Stocker, R.F. (2000). Metamorphosis in Drosophila and other insects: the fate of neurons throughout the stages. Progr. Neurobiol. 62, 89–111. Tix, S., Bate, M., and Technau, G.M. (1989). Pre-existing neuronal pathways in the leg imaginal discs of Drosophila. Development 107, 855–862. Tix, S., Minden, J.S., and Technau, G.M. (1989). Preexisting neuronal pathways in the developing optic lobes of Drosophila. Development 105, 739–746.

4332. Tjian, R. and Maniatis, T. (1994). Transcriptional activation: a complex puzzle with few easy pieces. Cell 77, 5–8. [See also 2001 references: Curr. Biol. 11, R510–R513; Science 293, 1054–1055.] 4333. Toba, G., Ohsako, T., Miyata, N., Ohtsuka, T., Seong, K.-H., and Aigaki, T. (1999). The gene search system: a method for efﬁcient detection and rapid molecular identiﬁcation of genes in Drosophila melanogaster. Genetics 151, 725–737. 4334. Tobler, H. (1966). Zellspeziﬁsche Determination und Beziehung zwischen Proliferation und Transdetermina¨ tion in Bein- und Flugelprimordien von Drosophila melanogaster. J. Embryol. Exp. Morph. 16, 609–633. 4335. Tobler, H. (1969). Beeinﬂussung der Borstendifferenzierung und Musterbildung durch Mitomycin bei Drosophila melanogaster. Experientia 25, 213–214. 4336. Tobler, H. and Huber, S. (1972). Effect of nitrogen mustard (HN-2) on the development of the male foreleg of Drosophila melanogaster. Dros. Info. Serv. 49, 99–100. 4337. Tobler, H. and Maier, V. (1970). Zur Wirkung von Senfgasl¨osungen auf die Differenzierung des Borstenorganes und auf die Transdeterminationsfrequenz bei Drosophila melanogaster. W. Roux’ Arch. 164, 303–312. 4338. Tobler, H., Rothenbuhler, V., and Nothiger, R. (1973). A study of the differentiation of bracts in Drosophila melanogaster using two mutations, H 2 and sv de . Experientia 29, 370–371. 4339. Toby, G., Law, S.F., and Golemis, E.A. (1998). Vectors to target protein domains to different cellular compartments. BioTechniques 24, 637–640. 4340. Toering, S.J. (2001). Cellular localization of Drosophila Sprouty protein. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a165. 4341. Toffoli, T. and Margolus, N. (1987). “Cellular Automata Machines: A New Environment for Modeling.” M.I.T. Pr., Cambridge, Mass. 4342. Toh, Y. (1985). Structure of campaniform sensilla on the haltere of Drosophila prepared by cryoﬁxation. J. Ultrastruct. Res. 93, 92–100. 4343. Tokunaga, C. (1961). The differentiation of a secondary sex comb under the inﬂuence of the gene engrailed in Drosophila melanogaster. Genetics 46, 157–176. 4344. Tokunaga, C. (1962). Cell lineage and differentiation on the male foreleg of Drosophila melanogaster. Dev. Biol. 4, 489–516. 4345. Tokunaga, C. (1966). Msc: Multiple sex comb. Dros. Info. Serv. 41, 57. 4346. Tokunaga, C. (1978). Genetic mosaic studies of pattern formation in Drosophila melanogaster, with special reference to the prepattern hypothesis. In “Genetic Mosaics and Cell Differentiation,” Results and Problems in Cell Differentiation, Vol. 9, (W.J. Gehring, ed.). Springer-Verlag: Berlin, pp. 157–204. 4347. Tokunaga, C. (1982). Curt Stern, 1902–1981, in memoriam. Jpn. J. Genet. 57, 459–466. 4348. Tokunaga, C. and Gerhart, J.C. (1976). The effect of growth and joint formation on bristle pattern in D. melanogaster. J. Exp. Zool. 198, 79–96. 4349. Tokunaga, C. and Stern, C. (1965). The developmental autonomy of extra sex combs in Drosophila melanogaster. Dev. Biol. 11, 50–81. 4350. Tokunaga, C. and Stern, C. (1969). Determination of bristle direction in Drosophila. Dev. Biol. 20, 411–425. 4351. Tolkunova, E.N., Fujioka, M., Kobayashi, M., Deka, D., and Jaynes, J.B. (1998). Two distinct types of repression domain in Engrailed: one interacts with the Groucho corepressor

REFERENCES

4352.

4353.

4354.

4355.

4356. 4357. 4358.

4359. 4360.

4361.

4362.

4363. 4364.

4365.

4366.

4367.

4368.

4369.

4370.

4371.

and is preferentially active on integrated target genes. Mol. Cell. Biol. 18, 2804–2814. Tolwinski, N.S. and Wieschaus, E. (2001). Armadillo nuclear import is regulated by cytoplasmic anchor Axin and nuclear anchor dTCF/Pan. Development 128, 2107–2117. Tomilin, A., Rem´enyi, A., Lins, K., Bak, H., Leidel, S., Vriend, G., Wilmanns, M., and Sch¨oler, H.R. (2000). Synergism with the coactivator OBF-1 (OCA-B, BOB-1) is mediated by a speciﬁc POU dimer conﬁguration. Cell 103, 853–864. Tomita, K., Ishibashi, M., Nakahara, K., Ang, S.-L., Nakanishi, S., Guillemot, F., and Kageyama, R. (1996). Mammalian hairy and Enhancer of Split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis. Neuron 16, 723–734. Tomlinson, A. (1985). The cellular dynamics of pattern formation in the eye of Drosophila. J. Embryol. Exp. Morph. 89, 313–331. Tomlinson, A. (1988). Cellular interactions in the developing Drosophila eye. Development 104, 183–193. Tomlinson, A. (1989). An eye to the main chance. Nature 340, 510–511. Tomlinson, A. (1989). Short-range positional signals in the developing Drosophila eye. Development 1989 Suppl., 59– 63. Tomlinson, A. (1991). sevenless and its bride: a marriage of molecules? Curr. Biol. 1, 132–134. Tomlinson, A., Bowtell, D.D.L., Hafen, E., and Rubin, G.M. (1987). Localization of the sevenless protein, a putative receptor for positional information, in the eye imaginal disc of Drosophila. Cell 51, 143–150. Tomlinson, A., Kimmel, B.E., and Rubin, G.M. (1988). rough, a Drosophila homeobox gene required in photoreceptors R2 and R5 for inductive interactions in the developing eye. Cell 55, 771–784. Tomlinson, A. and Ready, D.F. (1986). Sevenless: a cellspeciﬁc homeotic mutation of the Drosophila eye. Science 231, 400–402. Tomlinson, A. and Ready, D.F. (1987). Cell fate in the Drosophila ommatidium. Dev. Biol. 123, 264–275. Tomlinson, A. and Ready, D.F. (1987). Neuronal differentiation in the Drosophila ommatidium. Dev. Biol. 120, 366– 376. Tomlinson, A., Strapps, W.R., and Heemskerk, J. (1997). Linking Frizzled and Wnt signaling in Drosophila development. Development 124, 4515–4521. Tomlinson, A. and Struhl, G. (1999). Decoding vectorial information from a gradient: sequential roles of the receptors Frizzled and Notch in establishing planar polarity in the Drosophila eye. Development 126, 5725–5738. Tomlinson, A. and Struhl, G. (2001). Delta/Notch and Boss/Sevenless signals act combinatorially to specify the Drosophila R7 photoreceptor. Molec. Cell 7, 487–495. Tomoyasu, Y., Nakamura, M., and Ueno, N. (1998). Role of Dpp signalling in prepattern formation of the dorsocentral mechanosensory organ in Drosophila melanogaster. Development 125, 4215–4224. Tomoyasu, Y., Ueno, N., and Nakamura, M. (2000). The Decapentaplegic morphogen gradient regulates the notal wingless expression through induction of pannier and u-shaped in Drosophila. Mechs. Dev. 96, 37–49. Torres, M. and S´anchez, L. (1991). The sisterless-b function of the Drosophila gene scute is restricted to the stage when the X : A ratio determines the activity of Sex-lethal. Development 113, 715–722. Torres, R., Firestein, B.L., Dong, H., Staudinger, J., Olson,

425

4372.

4373.

4374.

4375.

4376.

4377. 4378. 4379. 4380.

4381.

4382.

4383.

4384.

4385. 4386.

4387.

4388.

E.N., Huganir, R.L., Bredt, D.S., Gale, N.W., and Yancopoulos, G.D. (1998). PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands. Neuron 21, 1453–1463. Torres-Vazquez, J., Park, S., Warrior, R., and Arora, K. (2001). The transcription factor Schnurri plays a dual role in mediating Dpp signaling during embryogenesis. Development 128, 1657–1670. Torres-Vazquez, J., Warrior, R., and Arora, K. (2000). schnurri is required for dpp-dependent patterning of the Drosophila wing. Dev. Biol. 227, 388–402. Townsley, F.M. and Bienz, M. (2000). Actin-dependent membrane association of a Drosophila epithelial APC protein and its effect on junctional Armadillo. Curr. Biol. 10, 1339–1348. Toyoda, H., Kinoshita-Toyoda, A., and Selleck, S.B. (2000). Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that toutvelu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo. J. Biol. Chem. 275 #4, 2269– 2275. ¨ Traas, J., Hulskamp, M., Gendreau, E., and H¨ofte, H. (1998). Endoreduplication and development: rule without dividing? Curr. Opin. Plant Biol. 1, 498–503. [See also 2001 Cell 105, 297–306.] Travers, A. (1999). An engine for nucleosome remodeling. Cell 96, 311–314. Travers, A.A. (1992). The reprogramming of transcriptional competence. Cell 69, 573–575. Travis, J. (1993). Cell biologists explore “tiny caves”. Science 262, 1208–1209. Treacy, M.N., Neilson, L.I., Turner, E.E., He, X., and Rosenfeld, M.G. (1992). Twin of I-POU: a two amino acid difference in the I-POU homeodomain distinguishes an activator from an inhibitor of transcription. Cell 68, 491– 505. Treier, M., Bohmann, D., and Mlodzik, M. (1995). JUN cooperates with the ETS domain protein Pointed to induce photoreceptor R7 fate in the Drosophila eye. Cell 83, 753– 760. Treier, M., Seufert, W., and Jentsch, S. (1992). Drosophila UbcD1 encodes a highly conserved ubiquitin-conjugating enzyme involved in selective protein degradation. EMBO J. 11, 367–372. Treisman, J., Harris, E., and Desplan, C. (1991). The paired box encodes a second DNA-binding domain in the Paired homeo domain protein. Genes Dev. 5, 594–604. Treisman, J., Harris, E., Wilson, D., and Desplan, C. (1992). The homeodomain: a new face for the helix-turn-helix? BioEssays 14, 145–150. Treisman, J.E. (1999). A conserved blueprint for the eye? BioEssays 21, 843–850. Treisman, J.E. (2001). Drosophila homologues of the transcriptional coactivation complex subunits TRAP240 and TRAP230 are required for identical processes in eye-antennal disc development. Development 128, 603– 615. Treisman, J.E. and Heberlein, U. (1998). Eye development in Drosophila: formation of the eye ﬁeld and control of differentiation. Curr. Top. Dev. Biol. 39, 119–158. [See also 2001 Mechs.Dev. 108, 13–27.] Treisman, J.E., Lai, Z.-C., and Rubin, G.M. (1995). shortsighted acts in the decapentaplegic pathway in Drosophila eye development and has homology to a mouse TGF-βresponsive gene. Development 121, 2835–2845.

426

4389. Treisman, J.E., Luk, A., Rubin, G.M., and Heberlein, U. (1997). eyelid antagonizes wingless signaling during Drosophila development and has homology to the Bright family of DNA-binding proteins. Genes Dev. 11, 1949– 1962. 4390. Treisman, J.E. and Rubin, G.M. (1995). wingless inhibits morphogenetic furrow movement in the Drosophila eye disc. Development 121, 3519–3527. 4391. Treisman, J.E. and Rubin, G.M. (1996). Targets of glass regulation in the Drosophila eye disc. Mechs. Dev. 56, 17–24. 4392. Treisman, R. (1995). Inside the MADS box. Nature 376, 468–469. 4393. Treisman, R. (1996). Regulation of transcription by MAP kinase cascades. Curr. Opin. Cell Biol. 8, 205–215. [See also 2001 Nature 411, 330–334.] 4394. Triezenberg, S.J., Kingsbury, R.C., and McKnight, S.L. (1988). Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev. 2, 718–729. 4395. Trott, R.L., Kalive, M., Paroush, Z., and Bidwai, A.P. (2001). Drosophila melanogaster casein kinase II interacts with and phosphorylates the basic helix-loop-helix proteins m5, m7, and m8 derived from the Enhancer of split Complex. J. Biol. Chem. 276 #3, 2159–2167. 4396. Trowbridge, I.S. (1993). Dynamin, SH3 domains and endocytosis. Curr. Biol. 3, 773–775. 4397. True, J.R., Edwards, K.A., Yamamoto, D., and Carroll, S.B. (1999). Drosophila wing melanin patterns form by veindependent elaboration of enzymatic prepatterns. Curr. Biol. 9, 1382–1391. 4398. Trujillo-Cen´oz, O. (1985). The eye: development, structure and neural connections. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology,” Vol. 6 (G.A. Kerkut and L.I. Gilbert, eds.). Pergamon Pr.: Oxford, pp. 171–223. 4399. Tsuda, L., Inoue, Y.H., Yoo, M.-A., Mizuno, M., Hata, M., Lim, Y.-M., Adachi-Yamada, T., Ryo, H., Masamune, Y., and Nishida, Y. (1993). A protein kinase similar to MAP kinase activator acts downstream of the Raf kinase in Drosophila. Cell 72, 407–414. 4400. Tsuda, M., Kamimura, K., Nakato, H., Archer, M., Staatz, W., Fox, B., Humphrey, M., Olson, S., Futch, T., Kaluza, V., Siegfried, E., Stam, L., and Selleck, S.B. (1999). The cellsurface proteoglycan Dally regulates Wingless signalling in Drosophila. Nature 400, 276–280. 4401. Tsuji, T., Sato, A., Hiratani, I., Taira, M., Saigo, K., and Kojima, T. (2000). Requirements of Lim1, a Drosophila LIMhomeobox gene, for normal leg and antennal development. Development 127, 4315–4323. 4402. Tsujimura, H. (2001). Neuronal programmed cell death is controlled by cell death genes, hid and reaper, in the reorganization of the CNS during Drosophila metamorphosis. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a260. 4403. Tsukazaki, T., Chiang, T.A., Davison, A.F., Attisano, L., and Wrana, J.L. (1998). SARA, a FYVE domain protein that recruits Smad2 to the TGFβ receptor. Cell 95, 779–791. 4404. Tsuneizumi, K., Nakayama, T., Kamoshida, Y., Kornberg, T.B., Christian, J.L., and Tabata, T. (1997). Daughters against dpp modulates dpp organizing activity in Drosophila wing development. Nature 389, 627–631. 4405. Tsunoda, S., Sierralta, J., Sun, Y., Bodner, R., Suzuki, E., Becker, A., Socolich, M., and Zuker, C.S. (1997). A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade. Nature 388, 243– 249.

REFERENCES

4406. Tsunoda, S., Sierralta, J., and Zuker, C.S. (1998). Speciﬁcity in signaling pathways: assembly into multimolecular signaling complexes. Curr. Opin. Gen. Dev. 8, 419–422. 4407. Tucker, J.B., Milner, M.J., Currie, D.A., Muir, J.W., Forrest, D.A., and Spencer, M.-J. (1986). Centrosomal microtubule-organizing centres and a switch in the control of protoﬁlament number for cell surface-associated microtubules during Drosophila wing morphogenesis. Eur. J. Cell Biol. 41, 279–289. 4408. Tun, T., Hamaguchi, Y., Matsunami, N., Furukawa, T., Honjo, T., and Kawaichi, M. (1994). Recognition sequence of a highly conserved DNA binding protein RBP-Jκ. Nucleic Acids Res. 22, 965–971. 4409. Turing, A.M. (1937). On computable numbers, with an application to the Entscheidungs problem. Proc. Lond. Math Soc., Ser. 2 42, 230–265. 4410. Turing, A.M. (1952). The chemical basis of morphogenesis. Phil. Trans. Roy. Soc. London, Ser. B 237, 37–72. 4411. Tweedie, S., Ng, H.-H., Barlow, A.L., Turner, B.M., Hendrich, B., and Bird, A. (1999). Vestiges of a DNA methylation system in Drosophila melanogaster? Nature Genet. 23, 389–390. 4412. Tyler, A. (1947). An auto-antibody concept of cell structure, growth and differentiation. Growth 10 (Suppl.), 7–19. 4413. Tyler, J.K. and Kadonaga, J.T. (1999). The “dark side” of chromatin remodeling: repressive effects on transcription. Cell 99, 443–446. 4414. Udvardy, A. (1999). Dividing the empire: boundary chromatin elements delimit the territory of enhancers. EMBO J. 18, 1–8. 4415. Ueda, R., Togashi, S., Takahisa, M., Tsurumura, S., Mikuni, M., Kondo, K., and Miyake, T. (1992). Sensory mother cell division is speciﬁcally affected in a Cyclin-A mutant of Drosophila melanogaster. EMBO J. 11, 2935–2939. 4416. Uemura, T., Oda, H., Kraut, R., Hayashi, S., Kataoka, Y., and Takeichi, M. (1996). Zygotic Drosophila E-cadherin expression is required for processes of dynamic epithelial cell rearrangement in the Drosophila embryo. Genes Dev. 10, 659–671. 4417. Uemura, T., Shepherd, S., Ackerman, L., Jan, L.Y., and Jan, Y.N. (1989). numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos. Cell 58, 349–360. 4418. Uemura, T., Shiomi, K., Togashi, S., and Takeichi, M. (1993). Mutation of twins encoding a regulator of protein phosphatase 2A leads to pattern duplication in Drosophila imaginal discs. Genes Dev. 7, 429–440. 4419. Ullman, K.S., Powers, M.A., and Forbes, D.J. (1997). Nuclear export receptors: from importin to exportin. Cell 90, 967–970. ¨ 4420. Ullrich, B., Ushkaryov, Y.A., and Sudhof, T.C. (1995). Cartography of neurexins: more than 1000 isoforms generated by alternative splicing and expressed in distinct subsets of neurons. Neuron 14, 497–507. 4421. Underwood, E.M., Turner, F.R., and Mahowald, A.P. (1980). Analysis of cell movements and fate mapping during early embryogenesis in Drosophila melanogaster. Dev. Biol. 74, 286–301. 4422. Urness, L.D. and Thummel, C.S. (1995). Molecular analysis of a steroid-induced regulatory hierarchy: the Drosophila E74A protein directly regulates L71-6 transcription. EMBO J. 14, 6239–6246. 4423. Ursprung, H. (1963). Development and genetics of patterns. Amer. Zool. 3, 71–86. 4424. Ursprung, H. (1972). The ﬁne structure of imaginal disks.

REFERENCES

4425.

4426.

4427.

4428.

4429.

4430.

4431.

4432.

4433.

4434.

4435.

4436.

4437.

4438.

4439.

4440.

In “The Biology of Imaginal Disks,” Results and Problems in Cell Differentiation, Vol. 5, (H. Ursprung and R. N¨othiger, eds.). Springer-Verlag: Berlin, pp. 93–107. Ursprung, H. and Hadorn, E. (1962). Weitere Unter¨ suchungen uber Musterbildung in Kombinaten aus ¨ teilweise dissoziierten Flugel-Imaginalscheiben von Drosophila melanogaster. Dev. Biol. 4, 40–66. Ursprung, H. and N¨othiger, R., eds. (1972). “The Biology of Imaginal Disks.” Results and Problems in Cell Differentiation, Vol. 5, Springer-Verlag, Berlin. Usui, K. and Kimura, K.-i. (1992). Sensory mother cells are selected from among mitotically quiescent cluster of cells in the wing disc of Drosophila. Development 116, 601– 610. Usui, K. and Kimura, K.-i. (1993). Sequential emergence of the evenly spaced microchaetes on the notum of Drosophila. Roux’s Arch. Dev. Biol. 203, 151–158. Usui, K. and Simpson, P. (2000). Cellular basis of the dynamic behavior of the imaginal thoracic discs during Drosophila metamorphosis. Dev. Biol. 225, 13–25. Usui, T., Shima, Y., Shimada, Y., Hirano, S., Burgess, R.W., Schwarz, T.L., Takeichi, M., and Uemura, T. (1999). Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity under the control of Frizzled. Cell 98, 585–595. [See also 2001 Curr. Biol. 11, 859–863.] Usui-Ishihara, A., Ghysen, A., and Kimura, K.-I. (1995). Peripheral axonal pathway and cleaning behavior are correlated in Drosophila microchaetes. Dev. Biol. 167, 398–401. Usui-Ishihara, A., Simpson, P., and Usui, K. (2000). Larval multidendrite neurons survive metamorphosis and participate in the formation of imaginal sensory axonal pathways in the notum of Drosophila. Dev. Biol. 225, 357– 369. Utley, R.T., Ikeda, K., Grant, P.A., Cˆot´e, J., Steger, D.J., Eberharter, A., John, S., and Workman, J.L. (1998). Transcriptional activators direct histone acetyltransferase complexes to nucleosomes. Nature 394, 498–502. Vachon, G., Cohen, B., Pfeiﬂe, C., McGufﬁn, M.E., Botas, J., and Cohen, S.M. (1992). Homeotic genes of the Bithorax Complex repress limb development in the abdomen of the Drosophila embryo through the target gene Distalless. Cell 71, 437–450. Vaessin, H., Brand, M., Jan, L.Y., and Jan, Y.N. (1994). daughterless is essential for neuronal precursor differentiation but not for initiation of neuronal precursor formation in Drosophila embryo. Development 120, 935–945. Vaessin, H., Grell, E., Wolff, E., Bier, E., Jan, L.Y., and Jan, Y.N. (1991). prospero is expressed in neuronal precursors and encodes a nuclear protein that is involved in the control of axonal outgrowth in Drosophila. Cell 67, 941–953. Vallis, Y., Wigge, P., Marks, B., Evans, P.R., and McMahon, H.T. (1999). Importance of the pleckstrin homology domain of dynamin in clathrin-mediated endocytosis. Curr. Biol. 9, 257–260. Van de Bor, V., Walther, R., and Giangrande, A. (2000). Some ﬂy sensory organs are gliogenic and require glide/gcm in a precursor that divides symmetrically and produces glial cells. Development 127, 3735–3743. van de Wetering, M., Cavallo, R., Dooijes, D., van Beest, M., van Es, J., Loureiro, J., Ypma, A., Hursh, D., Jones, T., Bejsovec, A., Peifer, M., Mortin, M., and Clevers, H. (1997). Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell 88, 789–799. van den Heuvel, M., Harryman-Samos, C., Klingensmith,

427

4441.

4442.

4443.

4444.

4445.

4446.

4447.

4448.

4449. 4450.

4451.

4452.

4453.

4454.

4455.

4456.

4457.

J., Perrimon, N., and Nusse, R. (1993). Mutations in the segment polarity genes wingless and porcupine impair secretion of the wingless protein. EMBO J. 12, 5293–5302. van den Heuvel, M. and Ingham, P.W. (1996). smoothened encodes a receptor-like serpentine protein required for hedgehog signalling. Nature 382, 547–551. van den Heuvel, M., Nusse, R., Johnston, P., and Lawrence, P.A. (1989). Distribution of the wingless gene product in Drosophila embryos: A protein involved in cell-cell communication. Cell 59, 739–749. van der Bliek, A.M. and Meyerowitz, E.M. (1991). Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular trafﬁc. Nature 351, 411– 414. van der Geer, P. and Pawson, T. (1995). The PTB domain: a new protein module implicated in signal transduction. Trends Biochem. Sci. 20, 277–280. van der Geer, P., Wiley, S., Lai, V.K.-M., Olivier, J.P., Gish, G.D., Stephens, R., Kaplan, D., Shoelson, S., and Pawson, T. (1995). A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides. Curr. Biol. 5, 404–412. van der Hoeven, F., Z´ak´any, J., and Duboule, D. (1996). Gene transpositions in the HoxD complex reveal a hierarchy of regulatory controls. Cell 85, 1025–1035. van der Straten, A., Rommel, C., Dickson, B., and Hafen, E. (1997). The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila. EMBO J. 16, 1961– 1969. van der Vlag, J., den Blaauwen, J.L., Sewalt, R.G.A.B., van Driel, R., and Otte, A.P. (2000). Transcriptional repression mediated by Polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators. J. Biol. Chem. 275 #1, 697–704. van der Voorn, L. and Ploegh, H.L. (1992). The WD-40 repeat. FEBS Letters 307, 131–134. van Dijk, M.A. and Murre, C. (1994). extradenticle raises the DNA binding speciﬁcity of homeotic selector gene products. Cell 78, 617–624. Van Doren, M., Bailey, A.M., Esnayra, J., Ede, K., and Posakony, J.W. (1994). Negative regulation of proneural gene activity: hairy is a direct transcriptional repressor of achaete. Genes Dev. 8, 2729–2742. Van Doren, M., Ellis, H.M., and Posakony, J.W. (1991). The Drosophila extramacrochaetae protein antagonizes sequence-speciﬁc DNA binding by daughterless/achaetescute protein complexes. Development 113, 245–255. Van Doren, M., Powell, P.A., Pasternak, D., Singson, A., and Posakony, J.W. (1992). Spatial regulation of proneural gene activity: auto- and cross-activation of achaete is antagonized by extramacrochaetae. Genes Dev. 6, 2592– 2605. van Holde, K. and Zlatanova, J. (1996). Chromatin architectural proteins and transcription factors: a structural connection. BioEssays 18, 697–700. van Leeuwen, F., Harryman Samos, C., and Nusse, R. (1994). Biological activity of soluble wingless protein in cultured Drosophila imaginal disc cells. Nature 368, 342– 344. van Meyel, D.J., O’Keefe, D.D., Jurata, L.W., Thor, S., Gill, G.N., and Thomas, J.B. (1999). Chip and Apterous physically interact to form a functional complex during Drosophila development. Molec. Cell 4, 259–265. van Meyel, D.J., O’Keefe, D.D., Thor, S., Jurata, L.W., Gill, G.N., and Thomas, J.B. (2000). Chip is an essential

428

4458.

4459.

4460. 4461.

4462. 4463.

4464.

4465.

4466.

4467.

4468.

4469.

4470.

4471.

4472.

4473.

4474.

REFERENCES

cofactor for Apterous in the regulation of axon guidance in Drosophila. Development 127, 1823–1831. Van Vactor, D., Jr., Krantz, D.E., Reinke, R., and Zipursky, S.L. (1988). Analysis of mutants in Chaoptin, a photoreceptor cell-speciﬁc glycoprotein in Drosophila, reveals its role in cellular morphogenesis. Cell 52, 281–290. Van Vactor, D.L., Jr., Cagan, R.L., Kr¨amer, H., and Zipursky, S.L. (1991). Induction in the developing compound eye of Drosophila: multiple mechanisms restrict R7 induction to a single retinal precursor cell. Cell 67, 1145–1155. Van Valen, L. (1962). A study of ﬂuctuating asymmetry. Evolution 16, 125–142. Vandendries, E.R., Johnson, D., and Reinke, R. (1996). orthodenticle is required for photoreceptor cell development in the Drosophila eye. Dev. Biol. 173, 243–255. Vandervorst, P. and Ghysen, A. (1980). Genetic control of sensory connections in Drosophila. Nature 286, 65–67. Varadarajan, S. and VijayRaghavan, K. (1999). scalloped functions in a regulatory loop with vestigial and wingless to pattern the Drosophila wing. Dev. Genes Evol. 209, 10– 17. Vashee, S., Melcher, K., Ding, W.V., Johnston, S.A., and Kodadek, T. (1998). Evidence for two modes of cooperative DNA binding in vivo that do not involve direct proteinprotein interactions. Curr. Biol. 8, 452–458. V¨assin, H., Bremer, K.A., Knust, E., and Campos-Ortega, J.A. (1987). The neurogenic gene Delta of Drosophila melanogaster is expressed in neurogenic territories and encodes a putative transmembrane protein with EGF-like repeats. EMBO J. 6, 3431–3440. V¨assin, H. and Campos-Ortega, J.A. (1987). Genetic analysis of Delta, a neurogenic gene of Drosophila melanogaster. Genetics 116, 433–445. V¨assin, H., Vielmetter, J., and Campos-Ortega, J.A. (1985). Genetic interactions in early neurogenesis of Drosophila melanogaster. J. Neurogenetics 2, 291–308. V´azquez, M., Moore, L., and Kennison, J.A. (1999). The trithorax group gene osa encodes an ARID-domain protein that genetically interacts with the Brahma chromatinremodeling factor to regulate transcription. Development 126, 733–742. Venkatesh, S. and Singh, R.N. (1984). Sensilla on the third antennal segment of Drosophila melanogaster Meigen (Diptera: Drosophilidae). Int. J. Insect Morphol. Embryol. 13, 51–63. Venkatesh, T.R., Hyatt, V., Korayan, A., Brindzei, N., Fermin, H., Mbogho, M., Shamloula, H., Lightowlers, Z.M.A., and Pimentel, A. (2001). rugose (rg) encodes a Drosophila A kinase anchor protein (DAKAP550) and functions in multiple signaling pathways. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a166. Venkatesh, T.R., Zipursky, S.L., and Benzer, S. (1985). Molecular analysis of the development of the compound eye in Drosophila. Trends Neurosci. 8, 251–257. Ventura, F., Doody, J., Liu, F., Wrana, J.L., and Massagu´e, J. (1994). Reconstitution and transphosphorylation of TGFβ receptor complexes. EMBO J. 13, 5581–5589. Verdi, J.M., Schmandt, R., Bashirullah, A., Jacob, S., Salvino, R., Craig, C.G., Amgen EST Program, Lipshitz, H.D., and McGlade, C.J. (1996). Mammalian NUMB is an evolutionarily conserved signaling adapter protein that speciﬁes cell fate. Curr. Biol. 6, 1134–1145. Verheyen, E.M. and Cooley, L. (1994). Proﬁlin mutations disrupt multiple actin-dependent processes dur-

4475.

4476.

4477.

4478. 4479.

4480.

4481.

4482.

4483.

4484.

4485.

4486. 4487. 4488. 4489.

4490. 4491.

4492.

4493.

ing Drosophila development. Development 120, 717– 728. Verheyen, E.M., Mirkovic, I., MacLean, S.J., Langmann, C., Andrews, B.C., and MacKinnon, C. (2001). The tissue polarity gene nemo carries out multiple roles in patterning during Drosophila development. Mechs. Dev. 101, 119– 132. Verma, I.M., Stevenson, J.K., Schwarz, E.M., Antwerp, D.V., and Miyamoto, S. (1995). Rel/NF-κB/IκB family: intimate tales of association and dissociation. Genes Dev. 9, 2723– 2735. Verrijzer, C.P., van Oosterhout, J.A.W.M., van Weperen, W.W., and van der Vliet, P.C. (1991). POU proteins bend DNA via the POU-speciﬁc domain. EMBO J. 10, 3007– 3014. Vervoort, M. (2000). hedgehog and wing development in Drosophila: a morphogen at work? BioEssays 22, 460–468. Vervoort, M., Crozatier, M., Valle, D., and Vincent, A. (1999). The COE transcription factor Collier is a mediator of short-range Hedgehog-induced patterning of the Drosophila wing. Curr. Biol. 9, 632–639. Vervoort, M., Dambly-Chaudi`ere, C., and Ghysen, A. (1997). Cell fate determination in Drosophila. Curr. Opin. Neurobiol. 7, 21–28. Vervoort, M., Zink, D., Pujol, N., Victoir, K., Dumont, N., Ghysen, A., and Dambly-Chaudi`ere, C. (1995). Genetic determinants of sense organ identity in Drosophila: regulatory interactions between cut and poxn. Development 121, 3111–3120. Vieira, A.V., Lamaze, C., and Schmid, S.L. (1996). Control of EGF receptor signaling by clathrin-mediated endocytosis. Science 274, 2086–2089. Vignali, M., Steger, D.J., Neely, K.E., and Workman, J.L. (2000). Distribution of acetylated histones resulting from Gal4-VP16 recruitment of SAGA and NuA4 complexes. EMBO J. 19, 2629–2640. Villano, J.L. and Katz, F.N. (1995). four-jointed is required for intermediate growth in the proximal-distal axis in Drosophila. Development 121, 2767–2777. Villares, R. and Cabrera, C.V. (1987). The achaete-scute gene complex of D. melanogaster: conserved domains in a subset of genes required for neurogenesis and their homology to myc. Cell 50, 415–424. Villee, C.A. (1942). The phenomenon of homeosis. Am. Nat. 76, 494–506. Vincent, J.-P. (1994). Morphogens dropping like ﬂies? Trends Genet. 10, 383–385. Vincent, J.-P. (1998). Compartment boundaries: where, why and how? Int. J. Dev. Biol. 42, 311–315. Vincent, J.-P. and Lawrence, P.A. (1994). Drosophila wingless sustains engrailed expression only in adjoining cells: evidence from mosaic embryos. Cell 77, 909–915. Vincent, J.-P. and Lawrence, P.A. (1994). It takes three to distalize. Nature 372, 132–133. Vincent, J.-P. and O’Farrell, P.H. (1992). The state of engrailed expression is not clonally transmitted during early Drosophila development. Cell 68, 923–931. Vincent, S., Ruberte, E., Grieder, N.C., Chen, C.-K., Haerry, T., Schuh, R., and Affolter, M. (1997). DPP controls tracheal cell migration along the dorsoventral body axis of the Drosophila embryo. Development 124, 2741– 2750. Vinson, C.R., Hai, T., and Boyd, S.M. (1993). Dimerization speciﬁcity of the leucine zipper-containing bZIP motif on

REFERENCES

4494.

4495. 4496. 4497. 4498.

4499.

4500. 4501. 4502.

4503.

4504.

4505.

4506.

4507.

4508. 4509. 4510. 4511. 4512. 4513.

4514. 4515. 4516.

4517.

DNA binding: prediction and rational design. Genes Dev. 7, 1047–1058. Vinson, C.R., Sigler, P.B., and McKnight, S.L. (1989). Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science 246, 911–916. Virshup, D.M. (2000). Protein phosphatase 2A: a panoply of enzymes. Curr. Opin. Cell Biol. 12, 180–185. Vogel, G. (1999). Many modes of transport for an embryo’s signals. Science 285, 1003–1005. Vogt, T.F. and Duboule, D. (1999). Antagonists go out on a limb. Cell 99, 563–566. Von Allmen, G., Hogga, I., Spierer, A., Karch, F., Bender, W., Gyurkovics, H., and Lewis, E. (1996). Splits in the fruitﬂy Hox gene complexes. Nature 380, 116. von Dassow, G., Meir, E., Munro, E.M., and Odell, G.M. (2000). The segment polarity network is a robust developmental module. Nature 406, 188–192. von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites. Nuc. Acids Res. 14, 4683–4690. von Heijne, G. (1995). Membrane protein assembly: rules of the game. BioEssays 17, 25–30. Von Ohlen, T. and Hooper, J.E. (1999). The ci D mutation encodes a chimeric protein whose activity is regulated by Wingless signaling. Dev. Biol. 208, 147–156. Von Ohlen, T., Lessing, D., Nusse, R., and Hooper, J.E. (1997). Hedgehog signaling regulates transcription through cubitus interruptus, a sequence-speciﬁc DNA binding protein. Proc. Natl. Acad. Sci. USA 94, 2404–2409. Vosshall, L.B., Amrein, H., Morozov, P.S., Rzhetsky, A., and Axel, R. (1999). A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96, 725–736. Vosshall, L.B., Price, J.L., Sehgal, A., Saez, L., and Young, M.W. (1994). Block in nuclear localization of period protein by a second clock mutation, timeless. Science 263, 1606– 1609. Vosshall, L.B., Wong, A.M., and Axel, R. (2000). An olfactory sensory map in the ﬂy brain. Cell 102, 147–159. [See also 2001 Nature 414, 204–208.] Vreezen, W.J. and Veldkamp, J.F. (1969). Selection and temperature effects on extra dorsocentral bristles in Drosophila melanogaster. Genetica 40, 19–39. Waddington, C.H. (1940). Genes as evocators in development. Growth 1 (Suppl.), 37–44. Waddington, C.H. (1940). The genetic control of wing development in Drosophila. J. Genet. 41, 75–139. Waddington, C.H. (1940). “Organizers and Genes.” Cambridge Univ. Pr., Cambridge. Waddington, C.H. (1941). Body-colour genes in Drosophila. Proc. Zool. Soc. Lond. Ser. A 111, 173–180. Waddington, C.H. (1943). The development of some “leg genes” in Drosophila. J. Genet. 45, 29–43. Waddington, C.H. (1957). “The Strategy of the Genes: A Discussion of Some Aspects of Theoretical Biology.” George Allen & Unwin, London. Waddington, C.H. (1960). Experiments on canalizing selection. Genet. Res. Camb. 1, 140–150. Waddington, C.H. (1962). “New Patterns in Genetics and Development.” Columbia Univ. Pr., New York. Waddington, C.H. (1966). Fields and gradients. In “Major Problems in Developmental Biology,” Symp. Soc. Dev. Biol., Vol. 25 (M. Locke, ed.). Acad. Pr.: New York, pp. 105–124. Waddington, C.H. (1973). The morphogenesis of patterns in Drosophila. In “Developmental Systems: Insects,” Vol.

429

4518.

4519.

4520.

4521.

4522.

4523.

4524.

4525.

4526.

4527.

4528.

4529. 4530. 4531.

4532.

4533.

4534.

4535. 4536.

2 (S.J. Counce and C.H. Waddington, eds.). Acad. Pr.: New York, pp. 499–535. Wade, P.A. and Wolffe, A.P. (1997). Chromatin: Histone acetyltransferases in control. Curr. Biol. 7, R82–R84. [See also 2001 BioEssays 23, 820–830.] Wagner, A. (1998). The fate of duplicated genes: loss or new function? BioEssays 20, 785–788. [See also 2001 references: BioEssays 23, 873–876; Trends Genet. 17, 237–239.] Wagner, G.P. and Altenberg, L. (1996). Complex adaptations and the evolution of evolvability. Evolution 50, 967– 976. Wagner, G.P., Chiu, C.-H., and Laubichler, M. (2000). Developmental evolution as a mechanistic science: the inference from developmental mechanisms to evolutionary processes. Amer. Zool. 40, 819–831. Wagner-Bernholz, J.T., Wilson, C., Gibson, G., Schuh, R., and Gehring, W.J. (1991). Identiﬁcation of target genes of the homeotic gene Antennapedia by enhancer detection. Genes Dev. 5, 2467–2480. Wai, P., Truong, B., and Bhat, K.M. (1999). Cell division genes promote asymmetric interaction between Numb and Notch in the Drosophila CNS. Development 126, 2759– 2770. Wainwright, S.M. and Ish-Horowicz, D. (1992). Point mutations in the Drosophila hairy gene demonstrate in vivo requirements for basic, helix-loop-helix, and WRPW domains. Mol. Cell. Biol. 12, 2475–2483. Wakimoto, B.T. and Kaufman, T.C. (1981). Analysis of larval segmentation in lethal genotypes associated with the Antennapedia gene complex in Drosophila melanogaster. Dev. Biol. 81, 51–64. Waksman, G., Shoelson, S.E., Pant, N., Cowburn, D., and Kuriyan, J. (1993). Binding of a high afﬁnity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms. Cell 72, 779–790. Walker, R.G., Willingham, A.T., and Zuker, C.S. (2000). A Drosophila mechanosensory transduction channel. Science 287, 2229–2234. ˜ Wall, M.A., Coleman, D.E., Lee, E., Iniguez-Lluhi, J.A., Posner, B.A., Gilman, A.G., and Sprang, S.R. (1995). The structure of the G protein heterotrimer Gia1 β1 γ2 . Cell 83, 1047–1058. Wallace, B. (1985). Reﬂections on the still-“hopeful monster.” Quart. Rev. Biol. 60, 31–42. Wallis, M. (1975). The molecular evolution of pituitary hormones. Biol. Rev. 50, 35–98. Walt, H. and Tobler, H. (1978). Ultrastructural analysis of differentiating bristle organs in wild-type, shavendepilate and Mitomycin C-treated larvae of Drosophila melanogaster. Biol. Cellulaire 32, 291–298. Walter, J., Dever, C.A., and Biggin, M.D. (1994). Two homeo domain proteins bind with similar speciﬁcity to a wide range of DNA sites in Drosophila embryos. Genes Dev. 8, 1678–1692. Waltzer, L. and Bienz, M. (1998). Drosophila CBP represses the transcription factor TCF to antagonize Wingless signalling. Nature 395, 521–525. Waltzer, L. and Bienz, M. (1999). A function of CBP as a transcriptional co-activator during Dpp signaling. EMBO J. 18, 1630–1641. Wandless, T.J. (1996). SH2 domains: a question of independence. Curr. Biol. 6, 125–127. Wang, G., Amanai, K., Wang, B., and Jiang, J. (2000). Interactions with Costal 2 and Suppressor of fused regulate

430

4537.

4538.

4539.

4540.

4541.

4542.

4543.

4544.

4545.

4546.

4547.

4548.

4549.

4550.

4551.

4552.

REFERENCES

nuclear translocation and activity of Cubitus interruptus. Genes Dev. 14, 2893–2905. Wang, G., Amanai, K., Wang, B., and Jiang, J. (2001). Interactions with Costal 2 and suppressor of fused regulate nuclear translocation and activity of Cubitus interruptus. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol. Addendum, 7. Wang, G., Wang, B., and Jiang, J. (1999). Protein kinase A antagonizes Hedgehog signaling by regulating both the activator and repressor forms of Cubitus interruptus. Genes Dev. 13, 2828–2837. Wang, Q.T. and Holmgren, R.A. (1999). The subcellular localization and activity of Drosophila Cubitus interruptus are regulated at multiple levels. Development 126, 5097– 5106. Wang, Q.T. and Holmgren, R.A. (2000). Nuclear import of Cubitus interruptus is regulated by Hedgehog via a mechanism distinct from Ci stabilization and Ci activation. Development 127, 3131–3139. Wang, S., Wang, Q., Crute, B.E., Melnikova, I.N., Keller, S.R., and Speck, N.A. (1993). Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor. Mol. Cell. Biol. 13, 3324– 3339. Wang, S., Younger-Shepherd, S., Jan, L.Y., and Jan, Y.N. (1997). Only a subset of the binary cell fate decisions mediated by Numb/Notch signaling in Drosophila sensory organ lineage requires Suppressor of Hairless. Development 124, 4435–4446. Wang, S.-H., Simcox, A., and Campbell, G. (2000). Dual role for Drosophila epidermal growth factor receptor signaling in early wing disc development. Genes Dev. 14, 2271–2276. [See also 2001 Genes Dev. 11, 470–475.] Wang, Y., Macke, J.P., Abella, B.S., Andreasson, K., Worley, P., Gilbert, D.J., Copeland, N.G., Jenkins, N.A., and Nathans, J. (1996). A large family of putative transmembrane receptors homologous to the product of the Drosophila tissue polarity gene frizzled. J. Biol. Chem. 271 #8, 4468–4476. Wang, Z. and Moran, M.F. (1996). Requirement for the adapter protein GRB2 in EGF receptor endocytosis. Science 272, 1935–1939. Wappner, P., Gabay, L., and Shilo, B.-Z. (1997). Interactions between the EGF receptor and DPP pathways establish distinct cell fates in the tracheal placodes. Development 124, 4707–4716. Ward, E.J. and Skeath, J.B. (2000). Characterization of a novel subset of cardiac cells and their progenitors in the Drosophila embryo. Development 127, 4959–4969. Ward, M.P., Mosher, J.T., and Crews, S.T. (1998). Regulation of bHLH-PAS protein subcellular localization during Drosophila embryogenesis. Development 125, 1599–1608. Warnock, D.E. and Schmid, S.L. (1996). Dynamin GTPase, a force-generating molecular switch. BioEssays 18, 885– 893. Warren, R. and Carroll, S. (1995). Homeotic genes and diversiﬁcation of the insect body plan. Curr. Opin. Gen. Dev. 5, 459–465. Warren, R.W. (1993). Deﬁning a neural “ground state” and photoreceptor cell identities in the Drosophila eye. BioEssays 15, 827–829. Warren, R.W., Nagy, L., Selegue, J., Gates, J., and Carroll, S. (1994). Evolution of homeotic gene regulation and function in ﬂies and butterﬂies. Nature 372, 458–461.

4553. Warrior, R. and Levine, M. (1990). Dose-dependent regulation of pair-rule stripes by gap proteins and the initiation of segment polarity. Development 110, 759–767. 4554. Wassarman, D.A., Solomon, N.M., Chang, H.C., Karim, F.D., Therrien, M., and Rubin, G.M. (1996). Protein phosphatase 2A positively and negatively regulates Ras1mediated photoreceptor development in Drosophila. Genes Dev. 10, 272–278. 4555. Wassarman, D.A., Therrien, M., and Rubin, G.M. (1995). The Ras signaling pathway in Drosophila. Curr. Opin. Gen. Dev. 5, 44–50. 4556. Wasserman, J.D. and Freeman, M. (1997). Control of EGF receptor activation in Drosophila. Trends Cell Biol. 7, 431– 436. 4557. Wasserman, J.D. and Freeman, M. (1998). An autoregulatory cascade of EGF receptor signaling patterns the Drosophila egg. Cell 95, 355–364. 4558. Wasserman, J.D., Urban, S., and Freeman, M. (2000). A family of rhomboid-like genes: Drosophila rhomboid-1 and roughoid/rhomboid-3 cooperate to activate EGF receptor signaling. Genes Dev. 14, 1651–1663. 4559. Wasylyk, B., Hahn, S.L., and Giovane, A. (1993). The Ets family of transcription factors. Eur. J. Biochem. 211, 7–18. 4560. Watson, J.D. (1970). “Molecular Biology of the Gene.” 2nd ed. W. A. Benjamin, New York. 4561. Watson, K.L., Justice, R.W., and Bryant, P.J. (1994). Drosophila in cancer research: the ﬁrst ﬁfty tumor suppressor genes. J. Cell Sci. 18 (suppl.), 19–33. 4562. Watts, C. (1997). Inside the gearbox of the dendritic cell. Nature 388, 724–725. 4563. Weatherbee, S.D. and Carroll, S.B. (1999). Selector genes and limb identity in arthropods and vertebrates. Cell 97, 283–286. 4564. Weatherbee, S.D., Halder, G., Kim, J., Hudson, A., and Carroll, S. (1998). Ultrabithorax regulates genes at several levels of the wing-patterning hierarchy to shape the development of the Drosophila haltere. Genes Dev. 12, 1474–1482. 4565. Weaver, T.A. and White, R.A.H. (1995). headcase, an imaginal speciﬁc gene required for adult morphogenesis in Drosophila melanogaster. Development 121, 4149–4160. 4566. Weber, U., Paricio, N., and Mlodzik, M. (2000). Jun mediates Frizzled-induced R3/R4 cell fate distinction and planar polarity determination in the Drosophila eye. Development 127, 3619–3629. 4567. Weber, U., Siegel, V., and Mlodzik, M. (1995). pipsqueak encodes a novel nuclear protein required downstream of seven-up for the development of photoreceptors R3 and R4. EMBO J. 14, 6247–6257. 4568. Wech, I., Bray, S., Delidakis, C., and Preiss, A. (1999). Distinct expression patterns of different Enhancer of split bHLH genes during embryogenesis of Drosophila melanogaster. Dev. Genes Evol. 209, 370–375. 4569. Wegner, M., Drolet, D.W., and Rosenfeld, M.G. (1993). POU-domain proteins: structure and function of developmental regulators. Curr. Opin. Cell Biol. 5, 488–498. 4570. Wehrli, M., Dougan, S.T., Caldwell, K., O’Keefe, L., Schwartz, S., Vaizel-Ohayon, D., Schejter, E., Tomlinson, A., and DiNardo, S. (2000). arrow encodes an LDLreceptor-related protein essential for Wingless signalling. Nature 407, 527–530. 4571. Wehrli, M., Rives, A., and DiNardo, S. (2001). How is the Wg signal transduced across the cell membrane? Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a155.

REFERENCES

4572. Wehrli, M. and Tomlinson, A. (1995). Epithelial planar polarity in the developing Drosophila eye. Development 121, 2451–2459. 4573. Wehrli, M. and Tomlinson, A. (1998). Independent regulation of anterior/posterior and equatorial/polar polarity in the Drosophila eye; evidence for the involvement of Wnt signaling in the equatorial/polar axis. Development 125, 1421–1432. 4574. Wei, X. and Ellis, H.M. (2001). Localization of the Drosophila MAGUK protein Polychaetoid is controlled by alternative splicing. Mechs. Dev. 100, 217–231. 4575. Weigmann, K. and Cohen, S.M. (1999). Lineage-tracing cells born in different domains along the PD axis of the developing Drosophila leg. Development 126, 3823–3830. 4576. Weigmann, K., Cohen, S.M., and Lehner, C.F. (1997). Cell cycle progression, growth and patterning in imaginal discs despite inhibition of cell division after inactivation of Drosophila Cdc2 kinase. Development 124, 3555–3563. 4577. Weihe, U., Milan, M., and Cohen, S.M. (2001). Posttranscriptional regulation of Apterous activity during wing development. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a188. [See also 2001 Development 128, 4615– 4622.] 4578. Weiner, J. (1999). “Time, Love, Memory.” Random House, New York. 4579. Weinkove, D. and Leevers, S.J. (2000). The genetic control of organ growth: insights from Drosophila. Curr. Opin. Gen. Dev. 10, 75–80. 4580. Weinmaster, G. (1997). The ins and outs of Notch signaling. Molec. Cell. Neurosci. 9, 91–102. 4581. Weinmaster, G. (1998). Notch signaling: direct or what? Curr. Opin. Gen. Dev. 8, 436–442. 4582. Weinmaster, G. (2000). Notch signal transduction: a real Rip and more. Curr. Opin. Gen. Dev. 10, 363–369. 4583. Weinstein, A. (1920). Homologous genes and linear linkage in Drosophila virilis. Proc. Natl. Acad. Sci. USA 6, 625– 639. 4584. Weintraub, H. (1993). The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell 75, 1241– 1244. 4585. Weis-Garcia, F. and Massagu´e, J. (1996). Complementation between kinase-defective and activation-defective TGF-β receptors reveals a novel form of receptor cooperativity essential for signaling. EMBO J. 15, 276–289. 4586. Weismann, A. (1864). Die nachembryonale Entwicklung der Musciden nach Beobachtungen an Musca vomitoria und Sarcophaga carnaria. Z. wiss. Zool. 14, 187–336. 4587. Weiss, A., Herzig, A., Jacobs, H., and Lehner, C.F. (1998). Continuous Cyclin E expression inhibits progression through endoreduplication cycles in Drosophila. Curr. Biol. 8, 239–242. 4588. Weiss, A. and Schlessinger, J. (1998). Switching signals on or off by receptor dimerization. Cell 94, 277–280. 4589. Weiss, P. (1939). “Principles of Development.” Henry Holt & Co., New York. 4590. Weiss, P. (1947). The problem of speciﬁcity in growth and development. Yale J. Biol. Med. 19, 235–278 (reprinted in Weiss’s 1968 book). 4591. Weiss, P. (1950). Perspectives in the ﬁeld of morphogenesis. Quart. Rev. Biol. 25, 177–198. 4592. Weiss, P. (1961). From cell to molecule. In “The Molecular Control of Cellular Activity,” (J.M. Allen, ed.). McGrawHill: New York, pp. 1–72 (reprinted in Weiss’s 1968 book).

431

4593. Weiss, P. (1969). The living system: determinism stratiﬁed. In “Beyond Reductionism: New Perspectives in the Life Sciences,” (A. Koestler and J.R. Smythies, eds.). MacMillan: New York, pp. 3–55. 4594. Weiss, P.A. (1968). “Dynamics of Development: Experiments and Inferences.” Acad. Pr., New York. 4595. Welte, M.A., Duncan, I., and Lindquist, S. (1995). The basis for a heat-induced developmental defect: deﬁning crucial lesions. Genes Dev. 9, 2240–2250. 4596. Wen, Y., Nguyen, D., Li, Y., and Lai, Z.-C. (2000). The N-terminal BTB/POZ domain and C-terminal sequences are essential for Tramtrack69 to specify cell fate in the developing Drosophila eye. Genetics 156, 195–203. 4597. Weng, G., Bhalla, U.S., and Iyengar, R. (1999). Complexity in biological signaling systems. Science 284, 92–96. 4598. Wente, S.R. (2000). Gatekeepers of the nucleus. Science 288, 1374–1377. 4599. Werb, Z. and Yan, Y. (1998). A cellular striptease act. Science 282, 1279–1280. [Cf. 2002 BioEssays 24, 8–12.] 4600. Wernet, M.F., Mollereau, B., and Desplan, C. (2001). Identifying genes involved in opsin regulation and photoreceptor cell type speciﬁcation using a Gal4 enhancer trap screen. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a270. 4601. Wesley, C.S. (1999). Notch and Wingless regulate expression of cuticle patterning genes. Mol. Cell. Biol. 19, 5743– 5758. 4602. Wesley, C.S. and Saez, L. (2000). Analysis of Notch lacking the carboxyl terminus identiﬁed in Drosophila embryos. J. Cell Biol. 149, 683–696. 4603. Wesley, C.S. and Saez, L. (2000). Notch responds differently to Delta and Wingless in cultured Drosophila cells. J. Biol. Chem. 275 #13, 9099–9101. 4604. Wessells, R.J., Grumbling, G., Donaldson, T., Wang, S.H., and Simcox, A. (1999). Tissue-speciﬁc regulation of Vein/EGF receptor signaling in Drosophila. Dev. Biol. 216, 243–259. 4605. West-Eberhard, M.J. (1989). Phenotypic plasticity and the origins of diversity. Annu. Rev. Ecol. Syst. 20, 249–278. 4606. Westheimer, F.H. (1987). Why nature chose phosphates. Science 235, 1173–1178. 4607. Whalen, J.H. and Simon, M.A. (2001). Genetic analysis of fat-head, a novel homeotic gene involved in eye and wing morphogenesis. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a189. 4608. Wharton, K., Ray, R.P., Findley, S.D., Duncan, H.E., and Gelbart, W.M. (1996). Molecular lesions associated with alleles of decapentaplegic identify residues necessary for TGF-β/BMP cell signaling in Drosophila melanogaster. Genetics 142, 493–505. 4609. Wharton, K.A. (1995). How many receptors does it take? BioEssays 17, 13–16. 4610. Wharton, K.A., Cook, J.M., Torres-Schumann, S., de Castro, K., Borod, E., and Phillips, D.A. (1999). Genetic analysis of the bone morphogenetic protein-related gene, gbb, identiﬁes multiple requirements during Drosophila development. Genetics 152, 629–640. 4611. Wharton, K.A., Johansen, K.M., Xu, T., and ArtavanisTsakonas, S. (1985). Nucleotide sequence from the neurogenic locus Notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 43, 567–581. 4612. Wharton, K.A., Jr., Franks, R.G., Kasai, Y., and Crews, S.T. (1994). Control of CNS midline transcription by

432

4613.

4614.

4615.

4616.

4617.

4618. 4619.

4620. 4621.

4622.

4623. 4624.

4625. 4626. 4627.

4628.

4629.

4630.

4631.

4632.

REFERENCES

asymmetric E-box-like elements: similarity to xenobiotic responsive regulation. Development 120, 3563–3569. Wharton, K.A., Ray, R.P., and Gelbart, W.M. (1993). An activity gradient of decapentaplegic is necessary for the speciﬁcation of dorsal pattern elements in the Drosophila embryo. Development 117, 807–822. Wharton, K.A., Thomsen, G.H., and Gelbart, W.M. (1991). Drosophila 60A gene, another transforming growth factor β family member, is closely related to human bone morphogenetic proteins. Proc. Natl. Acad. Sci. USA 88, 9214–9218. Wharton, K.A., Yedvobnick, B., Finnerty, V.G., and Artavanis-Tsakonas, S. (1985). opa: A novel family of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. melanogaster. Cell 40, 55–62. Wheeler, J.C., Shigesada, K., Gergen, J.P., and Ito, Y. (2000). Mechanisms of transcriptional regulation by Runt domain proteins. Sems. Cell Dev. Biol. 11, 369–375. Wheeler, S.R., Brown, S.J., Panganiban, G., and Skeath, J.B. (2001). The evolution of neural patterning and the achaete-scute complex. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a259. White, K. (2000). Cell death: Drosophila Apaf-1–no longer in the (d)Ark. Curr. Biol. 10, R167–R169. White, K., Grether, M.E., Abrams, J.M., Young, L., Farrell, K., and Steller, H. (1994). Genetic control of programmed cell death in Drosophila. Science 264, 677–683. White, K., Tahaoglu, E., and Steller, H. (1996). Cell killing by the Drosophila gene reaper. Science 271, 805–807. White, N.M. and Jarman, A.P. (2000). Drosophila Atonal controls photoreceptor R8-speciﬁc properties and modulates both Receptor Tyrosine Kinase and Hedgehog signalling. Development 127, 1681–1689. White, P., Aberle, H., and Vincent, J.-P. (1998). Signaling and adhesion activities of mammalian β-catenin and plakoglobin in Drosophila. J. Cell Biol. 140, 183–195. White, R. (1994). Homeotic genes seek partners. Curr. Biol. 4, 48–50. White, R.A.H. and Akam, M.E. (1985). Contrabithorax mutations cause inappropriate expression of Ultrabithorax products in Drosophila. Nature 318, 567–569. White, R.A.H. and Wilcox, M. (1984). Protein products of the bithorax complex in Drosophila. Cell 39, 163–171. White, R.A.H. and Wilcox, M. (1985). Distribution of Ultrabithorax proteins in Drosophila. EMBO J. 4, 2035–2043. White, R.A.H. and Wilcox, M. (1985). Regulation of the distribution of Ultrabithorax proteins in Drosophila. Nature 318, 563–567. White, R.H. (1961). Analysis of the development of the compound eye in the mosquito, Aedes aegypti. J. Exp. Zool. 148, 223–239. White, R.H. (1963). Evidence for the existence of a differentiation center in the developing eye of the mosquito. J. Exp. Zool. 152, 139–147. White, R.J. (1990). Cell type-speciﬁc enhancement in the Drosophila embryo by consensus homeodomain binding sites. BioEssays 12, 537–539. White-Cooper, H., Sch¨afer, M.A., Alphey, L.S., and Fuller, M.T. (1998). Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila. Development 125, 125–134. Whitelaw, E. and Martin, D.I.K. (2001). Retrotransposons

4633.

4634.

4635. 4636.

4637. 4638. 4639. 4640. 4641.

4642.

4643.

4644.

4645. 4646.

4647.

4648.

4649.

4650. 4651.

4652.

as epigenetic mediators of phenotypic variation in mammals. Nature Genet. 27, 361–365. Whitﬁeld, W.G.F., Gonzalez, C., Maldonado-Codina, G., and Glover, D.M. (1990). The A- and B-type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that deﬁne separable phases of the G2-M transition. EMBO J. 9, 2563–2572. Whiting, A.R. (1934). Eye colours in the parasitic wasp Habrobracon and their behaviour in multiple recessives and in mosaics. J. Genetics 29, 99–107. Whiting, M.F. and Wheeler, W.C. (1994). Insect homeotic transformation. Nature 368, 696. Whitlock, K.E. (1993). Development of Drosophila wing sensory neurons in mutants with missing or modiﬁed cell surface molecules. Development 117, 1251–1260. Whitman, M. (1997). Feedback from inhibitory SMADs. Nature 389, 549–551. Whitman, M. (1998). Smads and early developmental signaling by the TGFβ superfamily. Genes Dev. 12, 2445–2462. Whitten, J.M. (1969). Coordinated development in the ﬂy foot: sequential cuticle secretion. J. Morph. 127, 73–104. Whitten, J.M. (1972). Comparative anatomy of the tracheal system. Annu. Rev. Ent. 17, 373–402. Whitten, J.M. (1976). Some observations on cellular organization and pattern in ﬂies. In “The Insect Integument,” (H.R. Hepburn, ed.). Elsevier: New York, pp. 277–297. Whittle, J.R.S. (1976). Clonal analysis of a genetically caused duplication of the anterior wing in Drosophila melanogaster. Dev. Biol. 51, 257–268. Whittle, J.R.S. (1976). Mutations affecting the development of the wing. In “Insect Development,” Symp. Roy. Entomol. Soc. Lond., Vol. 8 (P.A. Lawrence, ed.). Wiley: New York, pp. 118–131. Whittle, J.R.S. (1983). Litany and creed in the genetic analysis of development. In “Development and Evolution,” Symp. Brit. Soc. Dev. Biol., Vol. 6 (B.C. Goodwin, N. Holder, and C.C. Wylie, eds.). Cambridge Univ. Pr.: Cambridge, pp. 59–74. Whittle, J.R.S. (1990). Pattern formation in imaginal discs. Seminars Cell Biol. 1, 241–252. Whittle, J.R.S. (1998). How is developmental stability sustained in the face of genetic variation? Int. J. Dev. Biol. 42, 495–499. Widelitz, R.B., Jiang, T.-X., Chen, C.-W.J., Stott, N.S., and Chuong, C.-M. (1999). Wnt-7a in feather morphogenesis: involvement of anterior-posterior asymmetry and proximal-distal elongation demonstrated with an in vitro reconstitution model. Development 126, 2577–2587. Wiersdorff, V., Lecuit, T., Cohen, S.M., and Mlodzik, M. (1996). Mad acts downstream of Dpp receptors, revealing a differential requirement for dpp signaling in initiation and propagation of morphogenesis in the Drosophia eye. Development 122, 2153–2162. Wieschaus, E. (1978). Cell lineage relationships in the Drosophila embryo. In “Genetic Mosaics and Cell Differentiation,” Results and Problems in Cell Differentiation, Vol. 9, (W.J. Gehring, ed.). Springer-Verlag: Berlin, pp. 97– 118. Wieschaus, E. (1996). Embryonic transcription and the control of developmental pathways. Genetics 142, 5–10. Wieschaus, E. and Gehring, W. (1976). Clonal analysis of primordial disc cells in the early embryo of Drosophila melanogaster. Dev. Biol. 50, 249–263. Wieschaus, E. and Gehring, W. (1976). Gynandromorph

REFERENCES

4653.

4654.

4655.

4656.

4657.

4658.

4659.

4660. 4661. 4662. 4663. 4664.

4665.

4666.

4667.

4668.

4669. 4670.

4671. 4672.

analysis of the thoracic disc primordia in Drosophila melanogaster. W. Roux’s Arch. 180, 31–46. ¨ ¨ Wieschaus, E., Nusslein-Volhard, C., and Jurgens, G. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. III. Zygotic loci on the Xchromosome and fourth chromosome. Roux’s Arch. Dev. Biol. 193, 296–307. Wieschaus, E., Perrimon, N., and Finkelstein, R. (1992). orthodenticle activity is required for the development of medial structures in the larval and adult epidermis of Drosophila. Development 115, 801–811. Wigge, P. and McMahon, H.T. (1998). The amphiphysin family of proteins and their role in endocytosis at the synapse. Trends Neur. Sci. 21, 339–344. Wigge, P., Vallis, Y., and McMahon, H.T. (1997). Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain. Curr. Biol. 7, 554–560. Wigglesworth, V.B. (1940). Local and general factors in the development of “pattern” in Rhodnius prolixus (Hemiptera). J. Exp. Zool. 17, 180–200. Wigglesworth, V.B. (1961). The epidermal cell. In “The Cell and the Organism,” (J.A. Ramsay and V.B. Wigglesworth, eds.). Cambridge Univ. Pr.: Cambridge, pp. 127–143. Wigglesworth, V.B. (1961). Insect polymorphism–a tentative synthesis. In “Insect Polymorphism,” Symp. Roy. Entomol. Soc. Lond., Vol. 1 (J.S. Kennedy, ed.). E. W. Classey: Faringdon, Oxon, U.K., pp. 103–113. Wigglesworth, V.B. (1964). Homeostasis in insect growth. Symp. Soc. Exp. Biol. 18, 265–281. Wigglesworth, V.B. (1972). “The Principles of Insect Physiology.” 7th ed. Chapman and Hall, London. Wigglesworth, V.B. (1973). Evolution of insect wings and ﬂight. Nature 246, 127–129. Wigglesworth, V.B. (1988). The control of pattern as seen in the integument of an insect. BioEssays 9, 23–27. Wilcox, M., DiAntonio, A., and Leptin, M. (1989). The function of PS integrins in Drosophila wing morphogenesis. Development 107, 891–897. Wilcox, M. and Smith, R.J. (1977). Regenerative interactions between Drosophila imaginal discs of different types. Dev. Biol. 60, 287–297. Wilder, E.L. and Perrimon, N. (1995). Dual functions of wingless in the Drosophila leg imaginal disc. Development 121, 477–488. Wilder, E.L. and Perrimon, N. (1996). Genes involved in postembryonic cell proliferation in Drosophila. In “Metamorphosis: Postembryonic Reprogramming of Gene Expression in Amphibian and Insect Cells,” (L.I. Gilbert, J.R. Tata, and B.G. Atkinson, eds.). Acad. Pr.: New York, pp. 363–400. Wilkin, M.B., Becker, M.N., Mulvey, D., Phan, I., Chao, A., Cooper, K., Chung, H.-J., Campbell, I.D., Baron, M., and MacIntyre, R. (2000). Drosophila Dumpy is a gigantic extracellular protein required to maintain tension at epidermal-cuticle attachment sites. Curr. Biol. 10, 559– 567. Wilkins, A.S. (1985). The limits of molecular biology. BioEssays 3, 3. Wilkins, A.S. (1989). Organizing the Drosophila posterior pattern: why has the fruit ﬂy made life so complicated for itself? BioEssays 11, 67–69. Wilkins, A.S. (1993). “Genetic Analysis of Animal Development.” 2nd ed. Wiley-Liss, New York. Wilkins, A.S. (1995). Singling out the tip cell of the

433

4673.

4674. 4675.

4676.

4677.

4678. 4679.

4680.

4681.

4682.

4683.

4684.

4685. 4686.

4687.

4688.

4689.

4690.

4691.

4692.

Malpighian tubules–lessons from neurogenesis. BioEssays 17, 199–202. Wilkins, A.S. (1995). What’s in a (biological) term? . . . Frequently, a great deal of ambiguity. BioEssays 17, 375– 377. Wilkins, A.S. (1997). Canalization: a molecular genetic perspective. BioEssays 19, 257–262. Wilkins, A.S. and Gubb, D. (1991). Pattern formation in the embryo and imaginal discs of Drosophila: what are the links? Dev. Biol. 145, 1–12. Willert, K., Brink, M., Wodarz, A., Varmus, H., and Nusse, R. (1997). Casein kinase 2 associates with and phosphorylates Dishevelled. EMBO J. 16, 3089–3096. Willert, K., Logan, C.Y., Arora, A., Fish, M., and Nusse, R. (1999). A Drosophila Axin homolog, Daxin, inhibits Wnt signaling. Development 126, 4165–4173. Willert, K. and Nusse, R. (1998). β-catenin: a key mediator of Wnt signaling. Curr. Opin. Gen. Dev. 8, 95–102. Willert, K., Shibamoto, S., and Nusse, R. (1999). Wntinduced dephosphorylation of Axin releases β-catenin from the Axin complex. Genes Dev. 13, 1768–1773. Williams, A.F. and Barclay, A.N. (1988). The immunoglobulin superfamily–domains for cell surface recognition. Annu. Rev. Immunol. 6, 381–405. Williams, J.A., Bell, J.B., and Carroll, S.B. (1991). Control of Drosophila wing and haltere development by the nuclear vestigial gene product. Genes Dev. 5, 2481–2495. Williams, J.A. and Carroll, S.B. (1993). The origin, patterning and evolution of insect appendages. BioEssays 15, 567– 577. Williams, J.A., Paddock, S.W., and Carroll, S.B. (1993). Pattern formation in a secondary ﬁeld: a hierarchy of regulatory genes subdivides the developing Drosophila wing disc into discrete subregions. Development 117, 571–584. Williams, J.A., Paddock, S.W., Vorwerk, K., and Carroll, S.B. (1994). Organization of wing formation and induction of a wing-patterning gene at the dorsal/ventral compartment boundary. Nature 368, 299–305. Williams, R.W. and Herrup, K. (1988). The control of neuron number. Annu. Rev. Neurosci. 11, 423–453. Willis, J.H. (1986). The paradigm of stage-speciﬁc gene sets in insect metamorphosis: time for revision! Arch. Insect Biochem. Physiol. Suppl. 1, 47–57. Wilson, C., Pearson, R.K., Bellen, H.J., O’Kane, C.J., Grossniklaus, U., and Gehring, W. (1989). P-element-mediated enhancer detection: an efﬁcient method for isolating and characterizing developmentally regulated genes in Drosophila. Genes Dev. 3, 1301–1313. Wilson, D., Sheng, G., Lecuit, T., Dostatni, N., and Desplan, C. (1993). Cooperative dimerization of Paired class homeo domains on DNA. Genes Dev. 7, 2120–2134. Wilson, D.S., Guenther, B., Desplan, C., and Kuriyan, J. (1995). High resolution crystal structure of a Paired (Pax) class cooperative homeodomain dimer on DNA. Cell 82, 709–719. Wilson, K.L., Zastrow, M.S., and Lee, K.K. (2001). Lamins and disease: insights into nuclear infrastructure. Cell 104, 647–650. Wilson, P.A. and Melton, D.A. (1994). Mesodermal patterning by an inducer gradient depends on secondary cell-cell communication. Curr. Biol. 4, 676–686. Wilson, T.G. (1980). Correlation of phenotypes of the apterous mutation in Drosophila melanogaster. Dev. Genet. 1, 195–204.

434

4693. Wilson, T.G. (1981). Expression of phenotypes in a temperature-sensitive allele of the apterous mutation in Drosophila melanogaster. Dev. Biol. 85, 425–433. 4694. Wilton, J.C. and Matthews, G.M. (1996). Polarised membrane trafﬁc in hepatocytes. BioEssays 18, 229–236. 4695. Wimmer, E.A., Cohen, S.M., J¨ackle, H., and Desplan, C. (1997). buttonhead does not contribute to a combinatorial code proposed for Drosophila head development. Development 124, 1509–1517. 4696. Winchester, G. (1996). The Morgan lineage. Curr. Biol. 6, 100–101. 4697. Winfree, A.T. (1980). “The Geometry of Biological Time.” Springer-Verlag, Berlin. 4698. Winfree, A.T. (1984). A continuity principle for regeneration. In “Pattern Formation: A Primer in Developmental Biology,” (G.M. Malacinski and S.V. Bryant, eds.). Macmillan: New York, pp. 103–124. 4699. Winfree, A.T., Winfree, E.M., and Seifert, H. (1985). Organizing centers in a cellular excitable medium. Physica 17D, 109–115. 4700. Winick, J., Abel, T., Leonard, M.W., Michelson, A.M., Chardon-Loriaux, I., Holmgren, R.A., Maniatis, T., and Engel, J.D. (1993). A GATA family transcription factor is expressed along the embryonic dorsoventral axis in Drosophila melanogaster. Development 119, 1055–1065. 4701. Winston, J.T., Strack, P., Beer-Romero, P., Chu, C.Y., Elledge, S.J., and Harper, J.W. (1999). The SCFβ-TRCP -ubiquitin ligase complex associates speciﬁcially with phosphorylated destruction motifs in IκBα and β-catenin and stimulates IκBα ubiquitination in vitro. Genes Dev. 13, 270– 283. 4702. Winter, C.G., Wang, B., Ballew, A., Royou, A., Karess, R., Axelrod, J.D., and Luo, L. (2001). Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. Cell 105, 81–91. 4703. Wisotzkey, R.G., Mehra, A., Sutherland, D.J., Dobens, L.L., Liu, X., Dohrmann, C., Attisano, L., and Raftery, L.A. (1998). Medea is a Drosophila Smad4 homolog that is differentially required to potentiate DPP responses. Development 125, 1433–1445. 4704. Wistow, G. (1993). Lens crystallins: gene recruitment and evolutionary dynamism. Trends Biochem. Sci. 18, 301–306. 4705. Wittinghofer, F. (1998). Caught in the act of the switch-on. Nature 394, 317–320. 4706. Wittwer, F., van der Straten, A., Keleman, K., Dickson, B.J., and Hafen, E. (2001). Lilliputian: an AF4/FMR2-related protein that controls cell identity and cell growth. Development 128, 791–800. 4707. Wodarz, A. (2000). Tumor suppressors: linking cell polarity and growth control. Curr. Biol. 10, R624–R626. 4708. Wodarz, A. and Nusse, R. (1998). Mechanisms of Wnt signaling in development. Annu. Rev. Cell Dev. Biol. 14, 59–88. 4709. Wodarz, A., Ramrath, A., Kuchinke, U., and Knust, E. (1999). Bazooka provides an apical cue for Inscuteable localization in Drosophila neuroblasts. Nature 402, 544–547. 4710. Wolfe, M.S., Xia, W., Ostaszewski, B.L., Diehl, T.S., Kimberly, W.T., and Selkoe, D.J. (1999). Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and g-secretase activity. Nature 398, 513–517. 4711. Wolfe, S.A., Nekludova, L., and Pabo, C.O. (1999). DNA recognition by Cys2 His2 zinc ﬁnger proteins. Annu. Rev. Biophys. Biomol. Struct. 3, 183–212. 4712. Wolff, T. and Ready, D.F. (1991). The beginning of pattern formation in the Drosophila compound eye: the morpho-

REFERENCES

4713.

4714.

4715.

4716.

4717. 4718. 4719. 4720. 4721.

4722. 4723.

4724. 4725. 4726.

4727.

4728. 4729. 4730. 4731.

4732. 4733. 4734.

genetic furrow and the second mitotic wave. Development 113, 841–850. Wolff, T. and Ready, D.F. (1991). Cell death in normal and rough eye mutants of Drosophila. Development 113, 825– 839. Wolff, T. and Ready, D.F. (1991). In search of a role for growth factors in Drosophila eye development. Sems. Dev. Biol. 2, 305–316. Wolff, T. and Ready, D.F. (1993). Pattern formation in the Drosophila retina. In “The Development of Drosophila melanogaster,” Vol. 2 (M. Bate and A. Martinez Arias, eds.). Cold Spring Harbor Lab. Pr.: Plainview, N. Y., pp. 1277– 1325. Wolff, T. and Rubin, G.M. (1998). strabismus, a novel gene that regulates tissue polarity and cell fate decisions in Drosophila. Development 125, 1149–1159. Wolffe, A.P. (1994). Architectural transcription factors. Science 264, 1100–1101. Wolffe, A.P. (1997). Sinful repression. Nature 387, 16–17. Wolffe, A.P., Khochbin, S., and Dimitrov, S. (1997). What do linker histones do in chromatin? BioEssays 19, 249–255. Wolfram, S. (1984). Cellular automata as models of complexity. Nature 311, 419–424. Wolfsberg, T.G., Primakoff, P., Myles, D.G., and White, J.M. (1995). ADAM, a novel family of membrane proteins containing A Disintegrin And Metalloprotease domain: multipotential functions in cell-cell and cell-matrix interactions. J. Cell Biol. 131, 275–278. Wolfsberg, T.G. and White, J.M. (1996). ADAMs in fertilization and development. Dev. Biol. 180, 389–401. Wolpert, L. (1968). The French Flag Problem: a contribution to the discussion on pattern development and regulation. In “Towards a Theoretical Biology. I. Prolegomena,” (C.H. Waddington, ed.). Aldine Pub. Co.: Chicago, pp. 125– 133. Wolpert, L. (1969). Positional information and the spatial pattern of cellular differentiation. J. Theor. Biol. 25, 1–47. Wolpert, L. (1971). Positional information and pattern formation. Curr. Top. Dev. Biol. 6, 183–224. Wolpert, L. (1974). Positional information and the development of pattern and form. In “Lectures on Mathematics in the Life Sciences,” Vol. 6 Am. Math. Soc.: Providence, Rhode Island, pp. 27–41. Wolpert, L. (1978). Cell position and cell lineage in pattern formation and regulation. In “Stem Cells and Tissue Homeostasis,” Brit. Soc. Cell Biol. Sympos., Vol. 2 (B. Lord, C.S. Potten, and R. Cole, eds.). Cambridge Univ. Pr.: Cambridge, pp. 29–47. Wolpert, L. (1978). Pattern formation in biological development. Sci. Am. 239 #4, 154–164. Wolpert, L. (1981). Positional information and pattern formation. Phil. Trans. Roy. Soc. Lond. B 295, 441–450. Wolpert, L. (1984). Molecular problems of positional information. BioEssays 1, 175–177. Wolpert, L. (1985). Gradients, position and pattern: a history. In “A History of Embryology,” Symp. Brit. Soc. Dev. Biol., Vol. 8 (T.J. Horder, J.A. Witkowski, and C.C. Wylie, eds.). Cambridge U. Pr.: New York, pp. 347–362. Wolpert, L. (1989). Positional information revisited. Development 1989 Suppl., 3–12. Wolpert, L. (1994). Positional information and pattern formation in development. Dev. Genet. 15, 485–490. Wolpert, L. (1996). One hundred years of positional information. Trends Genet. 12, 359–364.

REFERENCES

4735. Wolpert, L. and Lewis, J.H. (1975). Towards a theory of development. Fed. Proc. 34, 14–20. 4736. Wolpert, L. and Stein, W.D. (1984). Positional information and pattern formation. In “Pattern Formation: A Primer in Developmental Biology,” (G.M. Malacinski and S.V. Bryant, eds.). Macmillan: New York, pp. 3–21. 4737. Wolsky, A. and Wolsky, M.I. (1990). Constraints in the development and evolution of the arthropodan compound eye. In “Organizational Constraints on the Dynamics of Evolution,” (J. Maynard Smith and G. Vida, eds.). Manchester Univ. Pr.: Manchester, pp. 133–140. 4738. Wong, E.S.M., Lim, J., Low, B.C., Chen, Q., and Guy, G.R. (2001). Evidence for direct interaction between Sprouty and Cbl. J. Biol. Chem. 276 #8, 5866–5875. 4739. Wong, L.L. and Adler, P.N. (1993). Tissue polarity genes of Drosophila regulate the subcellular location for prehair initiation in pupal wing cells. J. Cell Biol. 123, 209–221. 4740. Wong, W.T., Schumacher, C., Salcini, A.E., Romano, A., Castagnino, P., Pelicci, P.G., and Di Fiore, P.P. (1995). A protein-binding domain, EH, identiﬁed in the receptor tyrosine kinase substrate Eps15 and conserved in evolution. Proc. Natl. Acad. Sci. USA 92, 9530–9534. 4741. Woodgett, J.R. (1991). A common denominator linking glycogen metabolism, nuclear oncogenes and development. Trends Biochem. Sci. 16, 177–181. 4742. Woodhouse, E., Hersperger, E., and Shearn, A. (1998). Growth, metastasis, and invasiveness of Drosophila tumors caused by mutations in speciﬁc tumor suppressor genes. Dev. Genes Evol. 207, 542–550. 4743. Woods, D.F. and Bryant, P.J. (1992). Genetic control of cell interactions in developing Drosophila epithelia. Annu. Rev. Genet. 26, 305–350. 4744. Woods, D.F. and Bryant, P.J. (1993). ZO-1, DlgA and PSD95/SAP90: homologous proteins in tight, septate and synaptic cell junctions. Mechs. Dev. 44, 85–89. 4745. Woods, D.F., Wu, J.-W., and Bryant, P.J. (1997). Localization of proteins to the apico-lateral junctions of Drosophila epithelia. Dev. Genet. 20, 111–118. 4746. Woods, R.E., Hercus, M.J., and Hoffmann, A.A. (1998). Estimating the heritability of ﬂuctuating asymmetry in ﬁeld Drosophila. Evolution 52, 816–824. 4747. Woods, R.E., Sgr`o, C.M., Hercus, M.J., and Hoffmann, A.A. (1999). The association between ﬂuctuating asymmetry, trait variability, trait heritability, and stress: a multiply replicated experiment on combined stresses in Drosophila melanogaster. Evolution 53, 493–505. 4748. Wootton, R. (1999). How ﬂies ﬂy. Nature 400, 112–113. 4749. Wootton, R.J. (1992). Functional morphology of insect wings. Annu. Rev. Entomol. 37, 113–140. 4750. Wootton, R.J. and Kukalov´a-Peck, J. (2000). Flight adaptations in Palaeozoic Palaeoptera (Insecta). Biol. Rev. 75, 129–167. 4751. Wrana, J. and Pawson, T. (1997). Mad about SMADs. Nature 388, 28–29. 4752. Wrana, J.L., Attisano, L., C´arcamo, J., Zentella, A., Doody, J., Laiho, M., Wang, X.-F., and Massagu´e, J. (1992). TGFβ signals through a heteromeric protein kinase receptor complex. Cell 71, 1003–1014. 4753. Wrana, J.L., Attisano, L., Wieser, R., Ventura, F., and Massagu´e, J. (1994). Mechanism of activation of the TGF-β receptor. Nature 370, 341–347. 4754. Wrana, J.L., Tran, H., Attisano, L., Arora, K., Childs, S.R., Massagu´e, J., and O’Connor, M.B. (1994). Two distinct transmembrane serine/threonine kinases from

435

4755. 4756.

4757.

4758.

4759.

4760.

4761.

4762.

4763.

4764.

4765.

4766.

4767.

4768.

4769. 4770.

4771.

Drosophila melanogaster form an activin receptor complex. Mol. Cell. Biol. 14, 944–950. Wray, G.A. (1998). Promoter logic. Science 279, 1871–1872. [See also 2001 Am. Sci. 89, 204–208.] Wreden, C., Verrotti, A.C., Schisa, J.A., Lieberfarb, M.E., and Strickland, S. (1997). Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA. Development 124, 3015–3023. Wright, J.W. and Copenhaver, P.F. (2000). Different isoforms of Fasciclin II play distinct roles in the guidance of neuronal migration during insect embryogenesis. Dev. Biol. 225, 59–78. Wright, J.W., Snyder, M.A., Schwinof, K.M., Combes, S., and Copenhaver, P.F. (1999). A role for fasciclin II in the guidance of neuronal migration. Development 126, 3217– 3228. Wu, C.-F., Berneking, J.M., and Barker, D.L. (1983). Acetylcholine synthesis and accumulation in the CNS of Drosophila larvae: Analysis of shibirets , a mutant with a temperature-sensitive block in synaptic transmission. J. Neurochem. 40, 1386–1396. Wu, J. and Cohen, S.M. (1999). Proximodistal axis formation in the Drosophila leg: subdivision into proximal and distal domains by Homothorax and Distal-less. Development 126, 109–117. Wu, J. and Cohen, S.M. (2000). Proximal distal axis formation in the Drosophila leg: distinct functions of Teashirt and Homothorax in the proximal leg. Mechs. Dev. 94, 47– 56. Wu, J.Y. and Rao, Y. (1999). Fringe: deﬁning borders by regulating the Notch pathway. Curr. Opin. Neurobiol. 9, 537–543. Wu, Q. and Maniatis, T. (1999). A striking organization of a large family of human cadherin-like cell adhesion genes. Cell 97, 779–790. Wu, X., Vakani, R., and Small, S. (1998). Two distinct mechanisms for differential positioning of gene expression borders involving the Drosophila gap protein giant. Development 125, 3765–3774. Wu, Z., Li, Q., Fortini, M.E., and Fischer, J.A. (1999). Genetic analysis of the role of the Drosophila fat facets gene in the ubiquitin pathway. Dev. Genet. 25, 312–320. ¨ Wulbeck, C. and Simpson, P. (2000). Expression of achaetescute homologues in discrete proneural clusters on the developing notum of the medﬂy Ceratitis capitata suggests a common origin for the stereotyped bristle patterns of higher Diptera. Development 127, 1411–1420. Wurmbach, E., Wech, I., and Preiss, A. (1999). The Enhancer of split complex of Drosophila melanogaster harbors three classes of Notch responsive genes. Mechs. Dev. 80, 171–180. Wurst, G., Hersperger, E., and Shearn, A. (1984). Genetic analysis of transdetermination in Drosophila. II. Transdetermination to wing of leg discs from a mutant which lacks wing discs. Dev. Biol. 106, 147–155. (N.B.: The “L6” gene studied here was renamed “vein.”) Wyman, R.J. (1986). Sequential induction and a homeotic switch of cell fate. Trends Neurosci. 9, 339–340. Xiao, B., Smerdon, S.J., Jones, D.H., Dodson, G.G., Soneji, Y., Aitken, A., and Gamblin, S.J. (1995). Structure of a 143-3 protein and implications for coordination of multiple signalling pathways. Nature 376, 188–191. Xie, T., Finelli, A.L., and Padgett, R.W. (1994). The

436

4772.

4773.

4774.

4775.

4776.

4777.

4778.

4779.

4780.

4781.

4782.

4783.

4784.

4785.

4786.

4787.

4788.

REFERENCES

Drosophila saxophone gene: a serine-threonine kinase receptor of the TGF-β superfamily. Science 263, 1756–1759. Xing, H., Kornfeld, K., and Muslin, A.J. (1997). The protein kinase KSR interacts with 14-3-3 protein and Raf. Curr. Biol. 7, 294–300. Xiong, W.-C. and Montell, C. (1993). tramtrack is a transcriptional repressor required for cell fate determination in the Drosophila eye. Genes Dev. 7, 1085–1096. Xiong, W.-C. and Montell, C. (1995). Defective glia induce neuronal apoptosis in the repo visual system of Drosophila. Neuron 14, 581–590. Xu, C., Kauffmann, R.C., Zhang, J., Kladny, S., and Carthew, R.W. (2000). Overlapping activators and repressors delimit transcriptional response to receptor tyrosine kinase signals in the Drosophila eye. Cell 103, 87–97. Xu, H.E., Rould, M.A., Xu, W., Epstein, J.A., Maas, R.L., and Pabo, C.O. (1999). Crystal structure of the human Pax 6 paired domain-DNA complex reveals speciﬁc roles for the linker region and carboxy-terminal subdomain in DNA binding. Genes Dev. 13, 1263–1275. Xu, P.-X., Zhang, X., Heaney, S., Yoon, A., Michelson, A.M., and Maas, R.L. (1999). Regulation of Pax6 expression is conserved between mice and ﬂies. Development 126, 383– 395. Xu, T. and Artavanis-Tsakonas, S. (1990). deltex, a locus interacting with the neurogenic genes, Notch, Delta and mastermind in Drosophila melanogaster. Genetics 126, 665–677. Xu, T., Caron, L.A., Fehon, R.G., and Artavanis-Tsakonas, S. (1992). The involvement of the Notch locus in Drosophila oogenesis. Development 115, 913–922. Xu, T., Rebay, I., Fleming, R.J., Scottgale, T.N., and Artavanis-Tsakonas, S. (1990). The Notch locus and the genetic circuitry involved in early Drosophila neurogenesis. Genes Dev. 4, 464–475. Xu, T. and Rubin, G.M. (1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223–1237. Xu, X., Yin, Z., Hudson, J.B., Ferguson, E.L., and Frasch, M. (1998). Smad proteins act in combination with synergistic and antagonistic regulators to target Dpp responses to the Drosophila mesoderm. Genes Dev. 12, 2354–2370. Xu, X.S., Kuspa, A., Fuller, D., Loomis, W.F., and Knecht, D.A. (1996). Cell-cell adhesion prevents mutant cells lacking myosin II from penetrating aggregation streams of Dictyostelium. Dev. Biol. 175, 218–226. Xu, Y.K. and Nusse, R. (1998). The Frizzled CRD domain is conserved in diverse proteins including several receptor tyrosine kinases. Curr. Biol. 8, R405–R406. Xue, L., Li, X., and Noll, M. (2001). Multiple protein functions of Paired in Drosophila development and their conservation in the Gooseberry and Pax 3 homologs. Development 128, 395–405. Xue, L. and Noll, M. (1996). The functional conservation of proteins in evolutionary alleles and the dominant role of enhancers in evolution. EMBO J. 15, 3722–3731. Yaffe, M.B., Rittinger, K., Volinia, S., Caron, P.R., Aitken, A., Leffers, H., Gamblin, S.J., Smerdon, S.J., and Cantley, L.C. (1997). The structural basis for 14-3-3:phosphopeptide binding speciﬁcity. Cell 91, 961–971. Yagi, Y., Suzuki, T., and Hayashi, S. (1998). Interaction between Drosophila EGF receptor and vnd determines three dorsoventral domains of the neuroectoderm. Development 125, 3625–3633.

4789. Yaich, L., Ooi, J., Park, M., Borg, J.-P., Landry, C., Bodmer, R., and Margolis, B. (1998). Functional analysis of the Numb phosphotyrosine-binding domain using sitedirected mutagenesis. J. Biol. Chem. 273 #17, 10381– 10388. 4790. Yamamoto, D. (1994). Signaling mechanisms in induction of the R7 photoreceptor in the developing Drosophila retina. BioEssays 16, 237–244. 4791. Yamamoto, D., Nihonmatsu, I., Matsuo, T., Miyamoto, H., Kondo, S., Hirata, K., and Ikegami, Y. (1996). Genetic interactions of pokkuri with seven in absentia, tramtrack and downstream components of the sevenless pathway in R7 photoreceptor induction in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 205, 215–224. 4792. Yamamoto, T., Harada, N., Kano, K., Taya, S.-i., Canaani, E., Matsuura, Y., Mizoguchi, A., Ide, C., and Kaibuchi, K. (1997). The Ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells. J. Cell Biol. 139, 785–795. 4793. Yamamoto, Y., Girard, F., Bello, B., Affolter, M., and Gehring, W.J. (1997). The cramped gene of Drosophila is a member of the Polycomb-group, and interacts with mus209, the gene encoding Proliferating Cell Nuclear Antigen. Development 124, 3385–3394. 4794. Yan, R., Luo, H., Darnell, J.E., Jr., and Dearolf, C.R. (1996). A JAK-STAT pathway regulates wing vein formation in Drosophila. Proc. Natl. Acad. Sci. USA 93, 5842–5847. 4795. Yanagawa, S., van Leeuwen, F., Wodarz, A., Klingensmith, J., and Nusse, R. (1995). The Dishevelled protein is modiﬁed by Wingless signaling in Drosophila. Genes Dev. 9, 1087–1097. 4796. Yanagawa, S.-i., Lee, J.-S., Haruna, T., Oda, H., Uemura, T., Takeichi, M., and Ishimoto, A. (1997). Accumulation of Armadillo induced by Wingless, Dishevelled, and dominantnegative Zeste-white 3 leads to elevated DE-cadherin in Drosophila clone 8 wing disc cells. J. Biol. Chem. 272 #40, 25243–25251. 4797. Yang, C.-H., Simon, M.A., and McNeill, H. (1999). mirror controls planar polarity and equator formation through repression of fringe expression and through control of cell afﬁnities. Development 126, 5857–5866. 4798. Yang, H.-P., Tanikawa, A.Y., and Kondrashov, A.S. (2001). Molecular nature of 11 spontaneous de novo mutations in Drosophila melanogaster. Genetics 157, 1285–1292. 4799. Yang, L. and Baker, N.E. (2001). Role of the EGFR/Ras/Raf pathway in speciﬁcation of photoreceptor cells in the Drosophila retina. Development 128, 1183–1191. 4800. Yang, S.-H., Whitmarsh, A.J., Davis, R.J., and Sharrocks, A.D. (1998). Differential targeting of MAP kinases to the ETS-domain transcription factor Elk-1. EMBO J. 17, 1740– 1749. 4801. Yang, S.-H., Yates, P.R., Whitmarsh, A.J., Davis, R.J., and Sharrocks, A.D. (1998). The Elk-1 ETS-domain transcription factor contains a mitogen-activated protein kinase targeting motif. Mol. Cell. Biol. 18, 710–720. 4802. Yang, W. and Cerione, R.A. (1999). Endocytosis: Is dynamin a “blue collar” or “white collar” worker? Curr. Biol. 9, R511– R514. 4803. Yang, X., Bahri, S., Klein, T., and Chia, W. (1997). Klumpfuss, a putative Drosophila zinc ﬁnger transcription factor, acts to differentiate between the identities of two secondary precursor cells within one neuroblast lineage. Genes Dev. 11, 1396–1408. 4804. Yang, X., Yeo, S., Dick, T., and Chia, W. (1993). The role

REFERENCES

4805.

4806.

4807.

4808.

4809.

4810.

4811.

4812.

4813.

4814.

4815.

4816.

4817.

4818.

4819.

of a Drosophila POU homeo domain gene in the speciﬁcation of neural precursor cell identity in the developing embryonic central nervous system. Genes Dev. 7, 504– 516. Yao, K.-M., Samson, M.-L., Reeves, R., and White, K. (1993). Gene elav of Drosophila melanogaster: a prototype for neuronal-speciﬁc RNA binding protein gene family that is conserved in ﬂies and humans. J. Neurobiol. 24, 723– 739. Yao, L.-C., Liaw, G.-J., Pai, C.-Y., and Sun, Y.H. (1999). A common mechanism for antenna-to-leg transformation in Drosophila: suppression of homothorax transcription by four HOM-C genes. Dev. Biol. 211, 268–276. Yap, A.S., Brieher, W.M., and Gumbiner, B.M. (1997). Molecular and functional analysis of cadherin-based adherens junctions. Annu. Rev. Cell Dev. Biol. 13, 119– 146. Yarnitzky, T., Min, L., and Volk, T. (1997). The Drosophila neuregulin homolog Vein mediates inductive interactions between myotubes and their epidermal attachment cells. Genes Dev. 11, 2691–2700. Ye, Y. and Fortini, M.E. (2000). Proteolysis and developmental signal transduction. Sems. Cell Dev. Biol. 11, 211– 221. Ye, Y., Lukinova, N., and Fortini, M.E. (1999). Neurogenic phenotypes and altered Notch processing in Drosophila Presenilin mutants. Nature 398, 525–529. Yeaman, C., Grindstaff, K.K., Hansen, M.D.H., and Nelson, W.J. (1999). Cell polarity: versatile scaffolds keep things in place. Curr. Biol. 9, R515–R517. Yedvobnick, B. and Kumar, A. (2001). Mastermind enhances the activation of a Notch pathway target in cell culture. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a260. [See also 2001 Genesis 30, 250–258.] Yedvobnick, B., Smoller, D., Young, P., and Mills, D. (1988). Molecular analysis of the neurogenic locus mastermind of Drosophila melanogaster. Genetics 118, 483–497. Yeh, E., Zhou, L., Rudzik, N., and Boulianne, G.L. (2000). Neuralized functions cell autonomously to regulate Drosophila sense organ development. EMBO J. 19, 4827–4837. [See also 2001 Curr. Biol., 11, 1675–1679.] Yenush, L., Fernandez, R., Myers, M.G., Jr., Grammer, T.C., Sun, X.J., Blenis, J., Pierce, J.H., Schlessinger, J., and White, M.F. (1996). The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis. Mol. Cell. Biol. 16, 2509–2517. Yeo, S.L., Lloyd, A., Kozak, K., Dinh, A., Dick, T., Yang, X., Sakonju, S., and Chia, W. (1995). On the functional overlap between two Drosophila POU homeo domain genes and the cell fate speciﬁcation of a CNS neural precursor. Genes Dev. 9, 1223–1236. Yin, J.C.P., Wallach, J.S., Wilder, E.L., Klingensmith, J., Dang, D., Perrimon, N., Zhou, H., Tully, T., and Quinn, W.G. (1995). A Drosophila CREB/CREM homolog encodes multiple isoforms, including a cyclic AMP-dependent protein kinase-responsive transcriptional activator and antagonist. Mol. Cell. Biol. 15, 5123–5130. Yin, Z., Xu, X.-L., and Frasch, M. (1997). Regulation of the Twist target gene tinman by modular cis-regulatory elements during early mesoderm development. Development 124, 4971–4982. Yoshida, H., Kunisada, T., Kusakabe, M., Nishikawa, S., and Nishikawa, S.-I. (1996). Distinct stages of melanocyte dif-

437

4820.

4821.

4822. 4823.

4824.

4825.

4826.

4827.

4828.

4829.

4830.

4831.

4832.

4833.

4834.

4835.

4836.

4837.

ferentiation revealed by analysis of nonuniform pigmentation patterns. Development 122, 1207–1214. Yost, C., Britton, J., Loo, L.W., Edgar, B., and Eisenman, R. (2001). The role of dMyc and dMad in endoreplication. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a86. Young, M.W. (1998). The molecular control of circadian behavioral rhythms and their entrainment in Drosophila. Annu. Rev. Biochem. 67, 135–152. Young, M.W. (2000). The tick-tock of the biological clock. Sci. Am. 282 #3, 64–71. Young, M.W. and Wesley, C.S. (1997). Diverse roles for the Notch receptor in the development of D. melanogaster. Persp. Dev. Neurobiol. 4, 345–355. Younger-Shepherd, S., Vaessin, H., Bier, E., Jan, L.Y., and Jan, Y.N. (1992). deadpan, an essential pan-neural gene encoding an HLH protein, acts as a denominator in Drosophila sex determination. Cell 70, 911–922. Younossi-Hartenstein, A., Tepass, U., and Hartenstein, V. (1993). Embryonic origin of the imaginal discs of the head of Drosophila melanogaster. Roux’s Arch. Dev. Biol. 203, 60–73. Yoxen, E. (1985). Form and strategy in biology: reﬂections on the career of C. H. Waddington. In “A History of Embryology,” 8th Symp. Brit. Soc. Dev. Biol., (T.J. Horder, J.A. Witkowski, and C.C. Wylie, eds.). Cambridge Univ. Pr.: Cambridge, pp. 309–329. Yu, F., Morin, X., Cai, Y., Yang, X., and Chia, W. (2000). Analysis of partner of inscuteable, a novel player of Drosophila asymmetric divisions, reveals two distinct steps in Inscuteable apical localization. Cell 100, 399–409. Yu, H., Rosen, M.K., Shin, T.B., Seidel-Dugan, C., Brugge, J.S., and Schreiber, S.L. (1992). Solution structure of the SH3 domain of Src and identiﬁcation of its ligand-binding site. Science 258, 1665–1668. Yu, H.-H., Huang, A.S., and Kolodkin, A.L. (2000). Semaphorin-1a acts in concert with the cell adhesion molecules fasciclin II and connectin to regulate axon fasciculation in Drosophila. Genetics 156, 723–731. Yu, K., Srinivasan, S., Shimmi, O., Biehs, B., Rashka, K., E., Kimelman, D., O’Connor, M.B., and Bier, E. (2000). Processing of the Drosophila Sog protein creates a novel BMP inhibitory activity. Development 127, 2143–2154. Yu, K., Sturtevant, M.A., Biehs, B., Franc¸ois, V., Padgett, R.W., Blackman, R.K., and Bier, E. (1996). The Drosophila decapentaplegic and short gastrulation genes function antagonistically during adult wing vein development. Development 122, 4033–4044. Yu, X. and Bienz, M. (1999). Ubiquitous expression of a Drosophila adenomatous polyposis coli homolog and its localization in cortical actin caps. Mechs. Dev. 84, 69–73. Yu, X., Waltzer, L., and Bienz, M. (1999). A new Drosophila APC homologue associated with adhesive zones of epithelial cells. Nature Cell Biol. 1, 144–151. Yu, Y. and Pick, L. (1995). Non-periodic cues generate seven ftz stripes in the Drosophila embryo. Mechs. Dev. 50, 163–175. Yuan, Y.P., Schultz, J., Mlodzik, M., and Bork, P. (1997). Secreted Fringe-like signaling molecules may be glycosyltransferases. Cell 88, 9–11. Yuh, C.-H., Bolouri, H., and Davidson, E.H. (1998). Genomic cis-regulatory logic: experimental and computational analysis of a sea urchin gene. Science 279, 1896– 1902. Yuh, C.-H., Bolouri, H., and Davidson, E.H. (2001).

438

4838.

4839.

4840. 4841.

4842.

4843.

4844.

4845.

4846.

4847.

4848.

4849.

4850.

4851.

4852.

4853.

4854.

4855.

REFERENCES

Cis-regulatory logic in the endo16 gene: switching from a speciﬁcation to a differentiation mode of control. Development 128, 617–629. Yuh, C.-H., Moore, J.G., and Davidson, E.H. (1996). Quantitative functional interrelations within the cis-regulatory system of the S. purpuratus Endo16 gene. Development 122, 4045–4056. Yun, U.J., Kim, S.Y., Liu, J., Adler, P.N., Bae, E., Kim, J., and Park, W.J. (1999). The Inturned protein of Drosophila melanogaster is a cytoplasmic protein located at the cell periphery in wing cells. Dev. Genet. 25, 297–305. Zacharuk, R.Y. (1980). Ultrastructure and function of insect chemosensilla. Annu. Rev. Entomol. 25, 27–47. Zacharuk, R.Y. (1985). Antennae and sensilla. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology,” Vol. 6 (G.A. Kerkut and L.I. Gilbert, eds.). Pergamon: New York, pp. 1–69. Zaffran, S. and Frasch, M. (2000). Barbu: an E(spl) m4/mαrelated gene that antagonizes Notch signaling and is required for the establishment of ommatidial polarity. Development 127, 1115–1130. Zak, N.B. and Shilo, B.-Z. (1992). Localization of DER and the pattern of cell divisions in wild-type and Ellipse eye imaginal discs. Dev. Biol. 149, 448–456. Z´ak´any, J., G´erard, M., Favier, B., and Duboule, D. (1997). Deletion of a HoxD enhancer induces transcriptional heterochrony leading to transposition of the sacrum. EMBO J. 16, 4393–4402. Zalokar, M., Erk, I., and Santamaria, P. (1980). Distribution of ring-X chromosomes in the blastoderm of gynandromorphic D. melanogaster. Cell 19, 133–141. Zappavigna, V., Sartori, D., and Mavilio, F. (1994). Speciﬁcity of HOX protein function depends on DNA-protein and protein-protein interactions, both mediated by the homeo domain. Genes Dev. 8, 732–744. Zaret, K. and Wolffe, A.P. (2001). Chromosomes and expression mechanisms: The post-genomic era of gene control. Curr. Opin. Gen. Dev. 11, 121–123. Zecca, M., Basler, K., and Struhl, G. (1995). Sequential organizing activities of engrailed, hedgehog, and decapentaplegic in the Drosophila wing. Development 121, 2265– 2278. Zecca, M., Basler, K., and Struhl, G. (1996). Direct and longrange action of a Wingless morphogen gradient. Cell 87, 833–844. Zeeman, E.C. (1974). Primary and secondary waves in developmental biology. In “Lectures on Mathematics in the Life Sciences,” Vol. 7 Am. Math. Soc.: Providence, Rhode Island, pp. 69–161. Zeidler, M.P., Perrimon, N., and Strutt, D.I. (1999). The four-jointed gene is required in the Drosophila eye for ommatidial polarity speciﬁcation. Curr. Biol. 9, 1363–1372. Zeidler, M.P., Perrimon, N., and Strutt, D.I. (2000). Multiple roles for four-jointed in planar polarity and limb patterning. Dev. Biol. 228, 181–196. [See also 2001 Development 128, 3533–3542.] Zeitlinger, J. and Bohmann, D. (1999). Thorax closure in Drosophila: involvement of Fos and the JNK pathway. Development 126, 3947–3956. Zeitlinger, J., Kockel, L., Peverali, F.A., Jackson, D.B., Mlodzik, M., and Bohmann, D. (1997). Defective dorsal closure and loss of epidermal decapentaplegic expression in Drosophila fos mutants. EMBO J. 16, 7393–7401. Zelhof, A.C., Ghbeish, N., Tsai, C., Evans, R.M., and

4856.

4857.

4858.

4859.

4860.

4861.

4862.

4863.

4864.

4865.

4866.

4867.

4868.

4869.

4870.

4871.

McKeown, M. (1997). A role for Ultraspiracle, the Drosophila RXR, in morphogenetic furrow movement and photoreceptor cluster formation. Development 124, 2499– 2506. Zelhof, A.C., Yao, T.-P., Chen, J.D., Evans, R.M., and McKeown, M. (1995). Seven-up inhibits Ultraspiraclebased signaling pathways in vitro and in vivo. Mol. Cell. Biol. 15, 6736–6745. Zelzer, E., Wappner, P., and Shilo, B.-Z. (1997). The PAS domain confers target gene speciﬁcity of Drosophila bHLH/PAS proteins. Genes Dev. 11, 2079–2089. Zeng, C., Justice, N.J., Abdelilah, S., Chan, Y.-M., Jan, L.Y., and Jan, Y.N. (1998). The Drosophila LIM-only gene, dLMO, is mutated in Beadex alleles and might represent an evolutionarily conserved function in appendage development. Proc. Natl. Acad. Sci. USA 95, 10637–10642. Zeng, C., Younger-Shepherd, S., Jan, L.Y., and Jan, Y.N. (1998). Delta and Serrate are redundant Notch ligands required for asymmetric cell divisions within the Drosophila sensory organ lineage. Genes Dev. 12, 1086–1091. Zeng, H., Qian, Z., Myers, M.P., and Rosbash, M. (1996). A light-entrainment mechanism for the Drosophila circadian clock. Nature 380, 129–135. Zeng, L., Fagotto, F., Zhang, T., Hsu, W., Vasicek, T.J., Perry III, W.L., Lee, J.J., Tilghman, S.M., Gumbiner, B.M., and Costantini, F. (1997). The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation. Cell 90, 181–192. Zeng, W., Andrew, D.J., Mathies, L.D., Horner, M.A., and Scott, M.P. (1993). Ectopic expression and function of the Antp and Scr homeotic genes: the N terminus of the homeodomain is critical to functional speciﬁcity. Development 118, 339–352. Zeng, W., Wharton, K.A., Jr., Mack, J.A., Wang, K., Gadbaw, M., Suyama, K., Klein, P.S., and Scott, M.P. (2000). naked cuticle encodes an inducible antagonist of Wnt signalling. Nature 403, 789–794. ¨ Zhang, C.-C., Muller, J., Hoch, M., J¨ackle, H., and Bienz, M. (1991). Target sequences for hunchback in a control region conferring Ultrabithorax expression boundaries. Development 113, 1171–1179. Zhang, H. and Levine, M. (1999). Groucho and dCtBP mediate separate pathways of transcriptional repression in the Drosophila embryo. Proc. Natl. Acad. Sci. USA 96, 535– 540. Zhang, H., Levine, M., and Ashe, H.L. (2001). Brinker is a sequence-speciﬁc transcriptional repressor in the Drosophila embryo. Genes Dev. 15, 261–266. [See also 2001 EMBO J. 20, 5725–5736.] Zhang, J. and Carthew, R.W. (1998). Interactions between Wingless and DFz2 during Drosophila wing development. Development 125, 3075–3085. Zhang, J. and Jacobson, A.G. (1993). Evidence that the border of the neural plate may be positioned by the interaction between signals that induce ventral and dorsal mesoderm. Dev. Dynamics 196, 79–90. Zhang, Q. and Lu, X. (2000). semang affects the development of a subset of cells in the Drosophila compound eye. Mechs. Dev. 95, 113–122. Zhang, Q., Zheng, Q., and Lu, X. (1999). A genetic screen for modiﬁers of Drosophila Src42A identiﬁes mutations in Egfr, rolled and a novel signaling gene. Genetics 151, 697–711. Zhang, Z., Murphy, A., Hu, J.C., and Kodadek, T. (1999).

REFERENCES

4872.

4873.

4874. 4875.

4876.

4877.

4878.

4879. 4880.

4881.

4882.

4883.

4884.

4885.

Genetic selection of short peptides that support protein oligomerization in vivo. Curr. Biol. 9, 417–420. Zhao, J.J., Lazzarini, R.A., and Pick, L. (1993). The mouse Hox-1.3 gene is functionally equivalent to the Drosophila Sex combs reduced gene. Genes Dev. 7, 343–354. Zheng, L., Zhang, J., and Carthew, R.W. (1995). frizzled regulates mirror-symmetric pattern formation in the Drosophila eye. Development 121, 3045–3055. Zheng, N. and Gierasch, L.M. (1996). Signal sequences: the same yet different. Cell 86, 849–852. Zhong, W., Feder, J.N., Jiang, M.-M., Jan, L.Y., and Jan, Y.N. (1996). Asymmetric localization of a mammalian numb homolog during mouse cortical neurogenesis. Neuron 17, 43–53. Zhong, W., Jiang, M.-M., Weinmaster, G., Jan, L.Y., and Jan, Y.N. (1997). Differential expression of mammalian Numb, Numblike and Notch1 suggests distinct roles during mouse cortical neurogenesis. Development 124, 1887– 1897. Zhou, J., Barolo, S., Szymanski, P., and Levine, M. (1996). The Fab-7 element of the bithorax complex attenuates enhancer-promoter interactions in the Drosophila embryo. Genes Dev. 10, 3195–3201. Zhou, M.-M., Ravichandran, K.S., Olejniczak, E.T., Petros, A.M., Meadows, R.P., Sattler, M., Harlan, J.E., Wade, W.S., Burakoff, S.J., and Fesik, S.W. (1995). Structure and ligand recognition of the phosphotyrosine binding domain of Shc. Nature 378, 584–592. Zhu, A. and Kuziora, M.A. (1996). Functional domains in the Deformed protein. Development 122, 1577–1587. Zhu, A.J. and Watt, F.M. (1999). β-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion. Development 126, 2285–2298. Zhu, A.J., Zheng, L., Suyama, K., Ho, K.S., and Scott, M.P. (2001). Hedgehog signaling moves Smoothened protein to the cell surface. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a155. Zhu, L. and Skoultchi, A.I. (2001). Coordinating cell proliferation and differentiation. Curr. Opin. Gen. Dev. 10, 91– 97. Zhu, L., Wilken, J., Phillips, N.B., Narendra, U., Chan, G., Stratton, S.M., Kent, S.B., and Weiss, M.A. (2000). Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc ﬁngers. Genes Dev. 14, 1750–1764. Zhuo, N., Tyler, D.M., Joglekar, S., Sultan, R., and Baker, N.E. (2001). Mutations affecting cell competition and growth. Proc. 42nd Ann. Drosophila Res. Conf. Abstracts Vol., a86. Ziemer, A., Tietze, K., Knust, E., and Campos-Ortega, J.A. (1988). Genetic analysis of Enhancer of split, a locus involved in neurogenesis in Drosophila melanogaster. Genetics 119, 63–74.

439

4886. Zilian, O., Frei, E., Burke, R., Brentrup, D., Gutjahr, T., Bryant, P.J., and Noll, M. (1999). double-time is identical to discs overgrown, which is required for cell survival, proliferation and growth arrest in Drosophila imaginal discs. Development 126, 5409–5420. 4887. Zill, S.N. and Seyfarth, E.-A. (1996). Exoskeletal sensors for walking. Sci. Am. 275 #1, 86–90. 4888. Zimmerman, J.E., Bui, Q.T., Liu, H., and Bonini, N.M. (2000). Molecular genetic analysis of Drosophila eyes absent mutants reveals an eye enhancer element. Genetics 154, 237–246. 4889. Zink, D. and Paro, R. (1995). Drosophila Polycomb-group regulated chromatin inhibits the accessibility of a transactivator to its target DNA. EMBO J. 14, 5660–5671. 4890. Zipursky, S.L. and Rubin, G.M. (1994). Determination of neuronal cell fate: lessons from the R7 neuron of Drosophila. Annu. Rev. Neurosci. 17, 373–397. 4891. Zollman, S., Godt, D., Priv´e, G.G., Couderc, J.-L., and Laski, F.A. (1994). The BTB domain, found primarily in zinc ﬁnger proteins, deﬁnes an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc. Natl. Acad. Sci. USA 91, 10717– 10721. 4892. Zorin, I.D., Gerasimova, T.I., and Corces, V.G. (1999). The lawc gene is a new member of the trithorax-group that affects the function of the gypsy insulator of Drosophila. Genetics 152, 1045–1055. 4893. Zrzavy, J. and Stys, P. (1995). Evolution of metamerism in arthropoda: developmental and morphological perspectives. Q. Rev. Biol. 70, 279–295. [See also 2001 Evol. Dev. 3, 332–342.] 4894. Zuker, C.S. (1994). On the evolution of eyes: would you like it simple or compound? Science 265, 742–743. 4895. Zuker, C.S., Cowman, A.F., and Rubin, G.M. (1985). Isolation and structure of a rhodopsin gene from D. melanogaster. Cell 40, 851–858. 4896. Zuker, C.S. and Ranganathan, R. (1999). The path to speciﬁcity. Science 283, 650–651. 4897. zur Lage, P., Jan, Y.N., and Jarman, A.P. (1997). Requirement for EGF receptor signalling in neural recruitment during formation of Drosophila chordotonal sense organ clusters. Curr. Biol. 7, 166–175. 4898. zur Lage, P. and Jarman, A.P. (1999). Antagonism of EGFR and Notch signalling in the reiterative recruitment of Drosophila adult chordotonal sense organ precursors. Development 126, 3149–3157. 4899. Zusman, S.B. and Wieschaus, E. (1987). A cell marker system and mosaic patterns during early embryonic development in Drosophila melanogaster. Genetics 115, 725–736. 4900. Zwahlen, C., Li, S.-C., Kay, L.E., Pawson, T., and FormanKay, J.D. (2000). Multiple modes of peptide recognition by the PTB domain of the cell fate determinant Numb. EMBO J. 19, 1505–1515.

Index

abbreviations, xiii, xiv. See also genes, particular abdomen, xi, 49, 84, 141, 239 sternites, 2, 30, 31, 56 tergites, 2, 30–32, 56, 62, 122, 151 Akam, Michael, 79, 84, 246, 247, 303 anatomy, 2, 32 antenna, 83, 199, 251 bilateral symmetry, 191, 193, 209 bristles, 5, 7, 30, 63 encoding, xi, 37, 41, 45, 83, 101, 129, 191, 213, 239, 246, 254, 255, 297 geometry, 31, 32, 99, 105, 128, 137, 189, 201, 211 gradients, 30, 65, 99 head, 41, 96, 199 leg, 65, 99, 127, 131 metameres, 76, 79, 84, 239, 243–247, 254 mirror-symmetry planes, 62, 97, 99, 136, 167, 188, 198–201, 209 periodicity, 77, 84, 136, 225, 303, 304 sclerites, 2, 86, 89, 245, 299 sexual dimorphism, 1, 56, 62–65, 72, 80 thorax, 41, 193 wing, 139, 153, 155, 159, 177 antenna anatomy, 83, 199, 251 axes. See under axes bipolar duality (vs. eye), 114, 169, 235, 302 circuitry, genetic, 205, 249–252, 300 compartments, 4, 199 duplication vs. regeneration, 96, 169, 242 evolution, 252 fate map, 199 gene expression patterns, 91, 193, 205, 251, 288–296, 302 homeosis to leg, 80–85, 129, 249–252 homology to leg, 83, 249, 251, 299 Hox gene irrelevance, 249 Hox gene misexpression in, 249, 251 identity (vs. eye), 169 identity (vs. leg), 83, 246, 249–254 part of eye disc, xiii, 96, 169, 199, 302 sensilla, 29, 191, 199, 246, 276 topology (vs. leg), 83, 199, 299 wiring of axons, 191

apoptosis, 63, 77, 80, 92, 99, 100, 105, 118, 119, 123, 128, 135, 139, 157, 160, 227–229 Aristotle, xi, xiii arthropods, 93, 99, 246, 300, 305 Ashburner, Michael, xii asymmetry bilateral, 56, 67, 87 bristle patterns, 31 cellular, 7, 11, 24, 274, 304 circuitry, 89, 107 eye D/V, 203–211 ﬂuctuating, 21 growth, 154 growth potential, 118–119 mitotic, 7, 11, 24 ommatidia, 209–211 symmetry-breaking, 47, 209–211, 304 wing A/P, 188, 301 wing D/V, 160, 164–167 Auerbach, Charlotte, 76 axes antenna D-V, 199 antenna proximal-distal, 83, 251 as reference lines, 84, 87, 89, 97, 101–105, 109, 151, 188, 203, 205 body and limb, 30, 31, 155, 239 bristle development, 30 cell apical-basal, 24, 87, 124, 146, 273, 293, 304 chordotonal, 27 diffusion along, 65 embryo A-P, 76, 79, 84, 87–90, 299 embryo D-V, 87–91, 170, 239 embryo left-right, 87 eye A-P, 201, 208–211, 215, 227, 233–236, 305 eye centrifugal, 229 eye D-V, 202–211, 227, 233, 236, 299 eye rhabdomeres, 199 interdependence, 115 larva, 205 leg A-P, 89, 112 leg A-P vs. wing A-P, 137 leg disc centrifugal, 129, 131 leg disc medial-lateral, 92 leg disc upper(stalk)-lower, 92

leg D-V, 61, 65, 67, 91, 97–99, 103, 109, 112, 115, 118, 119, 124–127, 132 leg proximal-distal, 29, 30, 61, 67, 80, 83, 91, 115–118, 127–135, 148, 166, 201, 251, 300 morphogen usage, 158, 249 notum A-P, 30, 31, 37, 68 optic lobe medial-lateral, 196 positional information, 81, 87 sternite A-P, 30 tarsus D-V, 30, 62 tergite A-P, 30 tergite D-V, 30 thorax A-P, 107, 132 thorax D-V, 205 wing A-P, 89, 137–156, 165, 167, 177, 301 wing A-P vs. D-V, 158–161, 165 wing centripetal, 125 wing crossveins, 189 wing D-V, 96, 136, 137, 146, 156–167, 170, 301 wing proximal-distal, 132, 151, 159, 171 Baker, Nicholas, 225 Baker, William, 202 Bang, Anne, 23 Basler, Konrad, 151, 157 Bateson, William, 99, 237 Becker, Hans, 202 Bender, Welcome, 305 Benzer, Seymour, 202, 225 Bernard, Francis, 201 Blair, Seth, xii, 173, 301 Bodenstein, Dietrich, xii, 76 boundaries. See also compartment boundaries absence, 93, 95, 105, 123, 155, 203 as axes, 84, 87, 89, 97, 101–105, 109, 188, 203, 205 as chains of adhering cells, 151, 173 as guidelines, 61–65, 163, 164 cis-enhancers for, 171, 245 clonal, 67, 201, 202 convergence/divergence (leg outgrowths), 100 creation of, 78, 159–161, 173, 203–207, 236, 302

441

442

boundaries (contd.) crossing of, 132, 136, 141, 148–154, 160, 173, 185, 201–204 deformations, 151 denoted by slash mark (vs. hyphen), xiii diffusion barriers, 125, 126, 143 discontinuities, 93 effects, 105, 115 eye equator, 198–211, 296 fuzzy, 128, 167, 193, 305 gene dosage, 52, 69 gene expression (on/off), 89, 121, 131, 136, 153, 157–160, 165, 173, 177, 186, 191, 203, 207, 209, 234, 235, 299, 303 gene expression at, 167, 171, 207, 209, 296 gene expression, complementary, 78, 129, 131, 134, 135 Hairy/Achaete, 63 idiosyncrasies, 166 insulating, 190 intersections, 46, 90, 101, 137, 157, 234, 301 kinks (natural), 112, 148 L 2&7 , 124 leg dAC/vAC, 100, 109, 111, 117, 119, 132 leg segment, 136 maintenance, 148–153, 166, 173, 273 male/female, 1–4, 52, 67 metamere, 79 midline, 59, 62, 63, 72, 77, 89, 99, 109, 113, 118, 131, 151, 164, 177, 262 models, 100–111, 115, 122, 130, 163, 165, 302 moving, 137, 229–234 mutant/wild-type, 47, 52 neural/non-neural, 151 offsets, 97, 99, 109, 148, 224, 225 parasegment, 78, 84, 90 peripodial membrane, 139 proneural clusters, 191 rewiring of control, 171 segment, 84 sharpening, 78, 81, 84, 114, 184 sharpness, 45, 167 shifts, 89, 111, 119–123, 145, 188–190, 288 smooth vs. ragged, 160, 173 straddling of, 153, 164 straightening of, 135, 148–153, 160, 173–174 wing margin, 142, 147, 157, 159, 173, 245, 301 Wingless/Engrailed, 78, 89, 90 yellow/brown, 2 zones, 111, 139, 302 bracts, 5, 7, 28–29, 61, 63, 67, 99 Bray, Sarah, 224 Brehme, Katherine, xii Bridges, Calvin, xii, 37, 299 bristle patterns. See also bristles alignment, 67, 68 ancestral, 62, 63, 135 ancient, 31 antineural gradients, 69 antineural mask, 69 antineural RNAs, 73 antineural stripes, 59, 61, 62, 69, 75 asymmetry, 56, 67 basitarsus, 56, 62 bristle density, 31, 49, 53–56, 73, 163, 187, 230

INDEX

bristle displacements, 37, 39, 43, 47, 49, 53, 68, 69, 163, 191, 229, 230 brushes, 62 cell-size dependence, 56, 68 conﬂuent lawns, 163, 229 constant vs. variable, 31, 56, 67 constellations, 32, 68 CS vs. MS bristles, 67 evolution, 31, 36, 62, 65, 69, 255 ﬁne-tuning, 50, 62–69, 75, 228 functions, 31, 62, 65 furry stripes, 59 general problem, 31 genetic control, 31–75, 190–194, 230, 278–284 geometry, 31, 56, 59 gradients, 62 growth-dependence, 39, 50, 57, 63, 67 heterochronic superposition, 56, 57, 67 indeterminacy, 56, 59, 67, 68 inhibitory ﬁelds, 50, 55–67, 71–75, 194, 195, 279 isotropic, 32 lattice, 32, 229, 230 leg, 56–62, 65, 67 macrochaetes, 190–194, 255 macrochaetes vs. microchaetes, 31, 32, 56, 67, 68 misalignment, 63 modules, 62, 63 mutant phenotypes, 278–284 natural variation, 31, 37, 55, 56 notum, 37, 41, 55–61, 65, 68, 190–194, 255 other species, 67, 69, 255 periodic, 67 precision, 31, 56, 59–62, 65, 67, 69 proneural clusters, 39, 41–75, 191–195, 225, 229 proneural competence, 49, 59, 69, 72, 75 proneural ﬁelds, 62, 68, 71, 72, 304 proneural gradients, 164, 191 proneural landscape, 69 proneural machinery, 190 proneural plateaus, 62 proneural potential, 67, 68, 69 proneural RNA motifs, 73 proneural spots, 59, 62, 71 proneural stripes, 59–63, 67–71, 75, 163, 280, 304 proneural subclusters, 43, 46, 47, 50, 53, 69 reconstitution, 67 rotation, 63, 65, 67, 106 rows, 31, 32, 56–62, 67, 68, 99 rows, alignment of, 61, 65, 67 rows, double (wing), 159, 189 rows, extra, 61, 68, 80, 187 rows, transverse (leg), 31, 62, 63, 65, 299 rows, triple (wing), 148, 159, 164, 189 scutellum, 49, 61 sex comb, 32, 36, 62–67, 106, 246 sex combs, extra, 80, 134, 135, 148, 248 sex dimorphisms, 56, 62, 63, 65 size-independence, 55, 62, 67 spacing, 31, 50, 55–68, 99, 159, 187, 224, 225, 279 sternites, 56 sternopleura, 56, 86 stochastic factors, 51, 72, 75, 201, 203, 299 symmetry, 62 tandem, 32, 65, 74 tergites, 31, 56

tufts, 23, 49, 52, 53, 59 variety, 32 vs. vein patterns, 175 wing margin, 65, 67, 159 bristles. See also bristle patterns; sensory organ precursor (SOP) abdominal, 55 anatomy, 5, 30, 63 bracted vs. bractless, 7, 65, 67, 99, 134 cell fate transformations, 7–28, 52, 271–275 cell lineage, 4, 5, 21, 27, 30, 44 central, 63 chemosensory, 5, 27, 45, 57, 62, 67, 99 CNS projections, 191 deformed, 23, 48 dendrite, 5, 23, 27 determination of type, 27, 63, 72 differentiation, 5, 11, 29, 36, 48 elongation, 29 evolution, 28, 29, 30 extra, xiv, 20, 31, 45, 49–55, 59–62, 68–75, 80, 124, 164, 166, 194, 229, 278–284 eye, 30, 57, 199, 209 function, 5 genetic control, 5–28, 271–277 grooves, 23, 29, 298 head, 31, 61 homologies, 63 inducers, 39, 50 inhibitors, 53, 56 leg, 5, 21, 27–31, 36, 56, 57, 61–67 lengths, 29–31, 39, 41, 56, 62, 65, 73 macrochaetes, xiii, 23, 31–62, 67–71, 75, 99, 190–195, 278–284 macrochaetes vs. microchaetes, 20, 29–30, 57, 68, 191 mechanosensory, 5, 27, 45, 57, 67, 99 microchaetes, xiii, 31–62, 67–71, 191, 278–284 migration, 63 misoriented, 65 missing, xiv, 20, 23, 31, 37, 45, 48–52, 71, 194, 278–284 mutant phenotypes, 271–277 notum, 29–31, 36, 39, 53–57, 62–68, 75 number, 31, 67 origin of glial cell, 5 orphan, 63 photosensitivity, 5 pigmentation, 5, 23, 30, 63, 65, 99 polarity, 29, 65, 67, 80, 205, 293 sex comb, 7, 30, 36, 72, 149 shaft-to-socket spectrum, 23 shapes, 99 size, 27, 29–30, 68 sternites, 30 sternopleura, 55, 56 tergites, 30, 48, 65, 68 thoracic, 4, 30 timing of determination, 27, 30, 63, 68, 72 timing of differentiation, 29, 39, 57, 67, 92 timing of radioinsensitivity, 39 transformation to sensilla, 28 types (MS vs. CS), 5, 27, 65, 67, 99, 159 vibrissae (head), 299 vs. photoreceptors, 225 vs. scales (lepidopteran), 29, 47, 61, 65, 67 vs. sensilla, 27–28

INDEX

wing blade (ectopic), 49, 61 wing margin, 5, 18, 27, 31, 48, 56, 143, 159–167, 189, 272 Britten, Roy, 244 Brody, Thomas, xiii Bryant, Peter, xii, 33, 80, 83, 93, 94, 139, 155 Bryant, Susan, 93 Cadigan, Kenneth, 158 Cagan, Ross, 212, 217, 227 Campbell, Gerard, 115 Carroll, Sean, 41 Castelli-Gair, James, 246, 247 Cavodeassi, Florencia, 203 cell adhesion, 65, 81, 87, 90, 92, 107, 132–136, 148–153, 160, 161, 173–177, 193, 204, 223, 257–261, 274, 281, 297 cell afﬁnities, 87, 90, 107, 132, 148–153, 160, 173, 174, 193, 204 cell behaviors, 63, 85, 93, 96, 107, 122, 123, 132, 153, 190, 203, 235, 252 cell competition, 105, 153 cell components cytoskeleton, 11, 257, 295 lysosomes, 27 microﬁlaments, 11, 29, 125 microtubules, 15, 109, 125 microvilli, 9, 87, 109, 179, 199 nuclear matrix, 249 nuts and bolts, 263 recycling, 300 rhabdomeres, 197, 199, 223, 224 ribosomes, 29 cell cortex, 7, 9, 11, 15, 24, 179, 273, 274 cell cycle. See under mitosis cell death, 63, 80, 87, 89, 95–97, 100, 113, 119, 135, 156, 174, 227–228, 299 cell instructions, 4, 5, 9, 18, 20, 93, 99, 101, 115, 173, 205, 215, 216, 217, 228, 254, 255. See also pattern formation: rules cell jostling, 90, 91, 111, 149, 151, 173 cell junctions, 20, 72, 92 adherens, 11, 75, 179, 180, 263, 294 making and breaking, 201, 304 cell lineage, 1–4. See also compartment boundaries adult vs. larva, 86 bracts, 5, 7, 28, 61, 63 bristles, 4, 5, 27, 30, 44 clonal analysis, 1–4, 86, 91, 99, 123, 201, 202 clone fragmentation, 91 clone fusions, 153 clone outlines, 3, 39, 67, 91, 201 clone overlaps, 91 clone roundness vs. raggedness, 132, 149–153, 160, 190, 201, 203 clone shapes, 133, 151, 153 compartments, 4, 44, 63, 85, 89, 99, 103, 104, 146, 150, 243, 245 compartments, function of, 87, 107, 148 compartments, regeneration of, 121 ﬂuid vs. stereotyped, 3, 5, 7, 90, 91, 111, 149, 151, 173 heart, 4, 9 indeterminate, 3, 4, 85, 86, 91, 111, 123, 136, 149, 202, 212, 252 Minute technique, 4, 91, 202 mixing, 105 muscle, 4, 9 nervous system, 4, 11

443

Proximity-vs.-Pedigree Rule, 4 sensilla, 1, 4 sex comb, 63 sibling rivalry, 9, 10 sternopleura, 86 strategies, 1 tracing, 3, 90 cell migration, 86, 91, 99, 258 cell movements, 61–67, 86-91, 131, 148–153, 177, 211, 230 cell packing, 53, 55, 122, 134, 149, 174, 208, 233, 304 cell polarity, 24, 29, 65, 67, 79, 80, 87, 131–136, 205, 211, 293, 304 cell psychology amnesia, 85, 249 antisocial behavior, 153 delirium, 107 how cells think, xii, 11, 83, 99, 111, 114, 124, 134, 136, 138, 143, 149, 154, 189, 190 memory, 10, 65, 81–84, 143, 158, 211, 239, 248, 251 myopia, 93, 94, 173 not goal-oriented, 255, 300 obedience to rules, 300. See also rules schizophrenia, 65, 107 sibling rivalry, 9, 10 sociability vs. introspection, 1–4, 85, 299 suicidal tendencies, 228 what cells know, xii, 83, 85, 158, 244, 249, 254, 255 cell rearrangements, 63–67, 89–91, 139, 174, 193, 209, 211, 304 cell recruitment, 29, 30, 87, 208, 212–220, 224, 227–229 cell shapes, 87, 99, 122, 124, 135, 151, 215, 233, 272, 273, 305 cell signaling, 1–4, 50, 72. See also circuitry; signaling pathways ampliﬁcation, 13, 47, 188, 209, 211, 286, 300 amplitude, 53, 74, 103, 215 amplitude effects, 113, 154 amplitude modulation, 145, 303 analog vs. digital responses, 182 anisotropic, 37, 59, 65, 75 apical, 44 as evocation, 304 attenuation, 27, 134, 189, 264 attenuation, signal-dependent, 147, 182 autocrine, 52, 151, 167, 188 community effect, 85 competence to respond, 29, 35, 39, 43, 45, 109, 111, 145, 148, 153, 155, 164, 175, 215, 304, 305 contact-limited, 139, 145, 153, 157, 185, 285, 293 contact-mediated, 47, 53, 72, 93, 103, 135, 164, 165, 212, 215 context-dependent, 217 deafness, enforced, 35, 107, 150–153, 173, 229, 231, 235 deafness, natural, 55, 61, 107, 113, 117, 121, 145, 148, 150, 163–167, 183, 187, 188, 207, 209, 227, 295 default states, 11, 59, 85, 169, 216, 227 diffusion-mediated, 39, 46–49, 52–55, 72, 75, 80–85, 98, 101, 105, 118, 123–126, 133, 137–141, 167, 169, 182, 183, 203–209

direct vs. signal relay, 139, 141, 156, 167, 186, 215 duration, 182, 303 ecdysone, 11, 217, 229 endocrine, 92, 217 endocytosis, 53 gating into pathways, 10 hijacking by heritable determinant, 10 information content, 216, 217 intercellular negotiation, 94, 95 juxtacrine, 180, 212, 224, 304 ligand-independent, 182 mitosis-dependent, 217 mufﬂers, 134 muteness, enforced, 107, 153, 209 muteness, natural, 103, 107, 145, 148, 163–166, 183, 187, 207 need for mitotic quiescence, 44, 234, 304 one-bit (Stop! or Go!) signals, 215–218, 304 paracrine, 92, 125, 133, 166, 167, 231, 295 perception modulation, 125, 134, 143, 147, 158, 188–190, 286, 291 PI coordinates, 93, 99, 103 potentiation, 166, 207 primers vs. boosters, 216 qualitative vs. quantitative, 35, 83, 179 rate, 9, 181, 287 rectiﬁcation, 147 resetting, 179, 182 rheostat vs. solenoid mode, 182, 303 second messengers, 141 sensitivity of reception, 230, 303 signal-to-noise ratio, 81, 124, 211, 228, 304 signal-to-receiver ratio, 60, 167, 286, 303 speciﬁcity, 179 strategies, xii, 1, 9 subthreshold, 36, 47, 68, 69, 117, 134, 174 transcytosis, 27, 124, 146, 167 trans-ingestion of receptor, 175 cell size, 29–30, 55–57, 65, 67, 92 cell sorting, 90, 134, 148–151 cell states. See also circuitry afﬁnities, 132, 148–153. See also cell afﬁnities as singularities, 158 automatic sequence of, 215, 229 axon projections, 55, 191 Boolean, 7–12, 18–23, 84–85 border vs. non-border, 149–153, 173, 205 cellular automata, 235 compartmental, 85, 89, 107, 145, 148–153, 158–163, 167, 173, 188, 202–208, 248 competence, 148, 217 created at interfaces, 87, 89, 107, 131–134, 142, 145, 153–155, 159, 160, 164, 177, 208–211, 234, 302, 303 default. See under circuitry dependent vs. independent, 89, 90, 117 determined vs. speciﬁed, 81, 90, 173, 247–249 differentiated, 81, 87, 224 eye vs. antenna, 169 ﬁrm vs. transitory biases, 90, 173, 218, 248, 252 heritable vs. not heritable, 89, 90, 132, 143, 246–252 implementation by gene hierarchies, 149, 174, 177, 303 inherited by discs from embryo, 107, 164 intrinsic vs. extrinsic, 1–4, 9, 90–91, 124, 143, 149, 217

444

cell states (contd.) leg dAC vs. vAC, 111, 113 leg proximal vs. distal, 134 proneural, 75. See also under bristle patterns quadrants in wing pouch, 158 qualitative vs. quantitative, 29–30, 139, 150, 246, 301 quantitative shifts in, 134, 135, 143, 147, 154–156, 189, 190 Ras-dependent diversity, 179 speciﬁed vs. determined, 90, 111 spectrum within eye MF, 205 switching, 7–12, 18–23, 52, 84–85, 101, 123, 132, 150, 182, 216–219, 223, 229, 242, 254, 261 switching, fast vs. slow, 287 vein identities, various, 154–157 vein vs. intervein, 141, 182, 187, 188, 259, 261, 303 wing vs. hinge, 139, 158, 172, 301, 302 wing vs. notum, 169, 171–173 cell types discrete nature of, 72, 246, 299 encoding of, 10, 213, 243, 246, 254 immiscible, 90, 107, 132–134, 148, 150 iteration, 217 laser ablation, 217 neural vs. non-neural, 20, 216, 218, 224 ommatidium, 208–209, 223 photoreceptors, 196, 197, 223, 253. See also photoreceptors sequential emergence, 216 signaler vs. receiver, 107, 145, 148, 163–166, 183, 187, 207 squamous, 87, 122 vein vs. intervein, 174–175 cells, cultured, 50, 61, 92, 217, 288, 303 Child, George, 39 circuit diagrams for bristle cell fates, 7, 25, 27 for bristle patterning, 35, 41, 43, 47, 71, 75, 193 for compartments, 89, 111, 142, 145, 163, 205, 207 for disc identity, 169, 239, 251 for disc initiation, 89 for embryo segmentation, 78 for gene regulation, 17, 19, 41, 43, 47, 71, 75, 78, 111, 112, 117, 121, 127, 131, 141, 142, 145, 163, 169, 172, 177, 193, 205, 207, 211, 233, 239, 251 for leg segmentation, 127, 131 for photoreceptor cell fates, 233 for protein networks, 15, 23, 71, 109, 179 for regeneration, 104, 121 for signal transduction, 9, 109, 179 for vein patterning, 177 symbols, 25 circuitry. See also cell signaling; cell states; circuit diagrams; circuits; codes; computation; computer metaphor; gene regulation; links; logic; mechanisms; uncoupling ampliﬁcation, 13, 47, 122, 188, 209, 211, 286, 300 analog to digital, 72, 81, 158, 209, 251 analog vs. digital, 11, 21, 79, 145, 160, 189, 208, 246, 247, 303 antagonism, 18, 21, 24, 25, 48–51, 61, 71–75, 114–121, 129, 134, 157, 161, 167,

INDEX

169, 175, 179–182, 188, 234, 273, 275, 281–284, 292, 295, 303 antagonism vs. cooperation, 117 antagonism, self-evoked, 161 antenna, 205, 249 antenna vs. leg, 249–252, 300 asymmetric, 107, 118–119, 164–167, 203–211 auto-activation, 47–51, 72, 91, 107, 171, 219 autocatalysis, 47, 50, 51, 69, 72 auto-repression, 18, 107, 132 biasing, 10, 45, 47, 52, 72, 90, 109, 132, 164, 174, 191, 207–211, 216, 218, 227, 229, 248–252, 304 biasing by Fringe, 164–167, 203–208 bipolar duality, 114, 167–173, 302 bistable seesaws, 25, 47, 106, 114–118, 170, 209, 299, 302 branched control, xiv, 51, 161, 165 buffering, 21, 71, 73, 291 canalization, 73, 174 cascades, xiv, 11, 20, 92, 139, 144, 174, 179, 199, 211–218, 224, 244, 249, 265, 297 cell shaping, 23 circuit breakers, 137, 145 clocks, circadian, 5, 263 competition, 9, 10, 21, 23, 46–53, 59, 68, 161, 209, 211, 227, 229 damping, 21, 47, 69, 72, 134, 163–167, 182, 235, 292, 304 default states, 7, 11, 47, 59, 65, 85, 148, 167–173, 197, 216, 227, 228, 251, 299 design ﬂaws, 228 design principles, cell level, 73, 270, 304 design principles, gene level, 19, 20, 73, 79, 91, 129, 142, 145, 148, 161, 173, 182, 207, 248, 270, 299 design principles, protein level, 71–73, 79, 161, 163, 260, 263, 270, 300, 303 design principles, tissue level, 57, 59, 62–67, 73, 87, 89, 107, 131–134, 142, 145, 153, 158–164, 177, 199, 228, 234, 235, 270, 302–305 devices, various, 270 discs vs. embryo, 86, 109, 113 dorsal discs vs. ventral discs, 171 driving factors, 25 dual control, 79, 92, 124, 183, 234, 254, 301 effective range, 69, 72, 73, 107, 119, 161, 209, 218, 304 evolution, xi, 10, 31, 36, 63, 69, 72, 77, 136, 158, 173, 186, 223, 239, 244–247, 255, 262, 288, 299, 302, 305 evolutionary relics, 36, 174 feedback loops, xiv, 248, 252, 299, 300, 302 feedback loops, negative, 164, 248, 291–295, 303 feedback loops, positive, 47–53, 72, 73, 79, 89, 121, 209, 211, 234, 248 ﬁdelity, 10, 21, 72, 136, 209, 304 ﬁne-tuning, 47, 50, 61–69, 75, 151, 161, 174–177, 228, 305 ﬂip-ﬂop, 25, 114 general problem, xii glitches, 138 ground states, 47, 53, 134, 135, 170, 171, 216, 243, 249 impedance of ligand diffusion, 125, 143, 146, 285

in parallel vs. in series, 9, 44, 148, 161, 164, 181, 183, 291 indeterminacy, 56 installation, 161 limiting factors, xiii, 11, 12, 21–25, 29, 38, 60, 69, 72, 92, 113, 160, 182 modules, 72, 179, 205, 208, 209, 305 mutual activation, 44, 48, 90, 91, 133, 171 mutual exclusivity, 114 mutual repression, 50, 56, 77, 134 neural networks, xiii, 255 noise, 17, 21, 51, 228 on-then-off switching, 48, 91, 184 oogenesis, 170, 179, 181, 289 optimization, 79 orchestration, xii, 79, 133 overreaction, 63 overrides, 10, 91, 107, 119, 128, 133, 137, 145, 164, 171, 190, 193, 246, 248 physiological range, 50, 125, 132, 144, 151, 253 plasticity, 91 positive vs. negative control, 69, 79 precision, 45, 46, 73, 81, 211, 304, 305 priming, 48 protein-level, 72, 161, 263 quantitative-to-qualitative, 81 quirks, 85, 123, 132, 133, 161, 182, 235 rate control, 30, 68, 72, 73, 208, 229, 234 reconﬁguration, 255 rectiﬁers, 147 rewiring, 63, 171, 245, 255 rheostats vs. switches, 30, 156, 182, 303 robustness, 21, 73, 79, 81, 91, 98, 106, 136, 145, 174, 299, 304 safety switches, 145 saturation, 53, 72, 160, 167 scalar vs. vector, 72, 81, 209, 211 segmentation genes, 78, 79 sensing of amplitude, 147 sensing of cell number, 145 sensing of concentration, 81, 83, 158, 209, 211 sensing of polarity, 81, 131, 136, 209–211, 293 sensing of position, 81, 83, 142, 158, 177, 211, 212, 251 sensing of size, 81, 124 sensing of slope, 81, 92, 158, 205, 227 short-circuiting, xiv, 15, 157, 167, 195, 288 simulations, 79 slack, 73 solid-state, 300 SOP selection, 73, 75 stability vs. instability, 47, 51, 170, 174, 211, 228, 237 starting conditions, 236 steady-state, 90 stoichiometry, 13, 21, 61, 73, 163, 165, 171, 175, 286, 287, 291, 295, 298, 300, 303 superimposing, 10, 69 symbols, xiii, 11, 25 symmetry-breaking, 51, 148 synergy, 52, 71, 75, 157, 182, 185, 191, 252–254 system properties, 57, 73, 79, 90, 299 temporal control, 112, 121, 123, 129, 133–137, 141, 145, 161–167, 177, 196, 205, 208–209, 218, 233, 247 thresholds, 21, 23, 39, 43–51, 69–73, 81, 119, 128, 133, 134, 160, 167, 169, 183,

INDEX

193, 218, 228, 243, 247, 295, 297. See also under positional information time constraints, 10, 29, 41, 77, 154, 156 toggling of activator-repressor modes, 19, 61, 107, 139, 255, 288 toggling of Delta-Serrate modes, 163, 165, 207 tracking, 79, 91, 124, 183, 190 transduction, 20, 27, 35, 72, 81, 134, 285–296 triggers, 43, 47, 51, 72, 115–119, 155, 157, 169, 207, 217, 297 triggers, scalar vs. temporal, 11, 133, 215 virtuosity, 72, 79 wing veins vs. tracheal branching, 174 wing vs. notum, 87, 167–173, 190–193 wiring, cis-trans, 136 circuits, versatile Dpp-Wg antagonism, eye disc, 234 Dpp-Wg antagonism, leg disc, 104, 106, 112–115 Dpp-Wg cooperation, leg disc, 115–118 Dpp-Wg cooperation, notum, 190–193 Dpp-Wg cooperation, wing disc, 157–158, 189–190 Hh-Dpp, antenna, 205, 249 Hh-Dpp, leg disc, 105–111, 128–129, 249 Hh-Dpp, wing disc, 111, 137–145 Hh-Dpp-Wg, eye MF initiation, 234–236 Hh-Dpp-Wg, eye MF movement, 229–234 Hh-Wg, antenna, 205, 249 Hh-Wg, embryo, 87–91 Hh-Wg, leg disc, 105–111, 128–129, 249 Notch, bristle differentiation, 9–15, 25 Notch, bristle spacing, 47–53, 59, 75, 229 Notch, eye equator, 203–209 Notch, leg segmentation, 127, 135–136 Notch, ommatidial chirality, 209–211 Notch, photoreceptor identity, 216 Notch, R8p spacing, 227 Notch, wing margin, 161–167 Notch, wing veins, 175–177 Notch-EGFR, chordotonal organs, 74 Notch-EGFR, eye-antenna bipolar duality, 169, 235, 302 Notch-Wg, leg bipolar duality, 114 Notch-Wg, wing margin, 161–167 PCP (Wnt?), cell polarity (in general), 293 PCP (Wnt?), chirality of ommatidia, 209–211 PCP (Wnt?), polarity of leg joints, 131, 136 codes. See also circuitry abstract, 89, 243 amino acid, 257 area, 81, 85, 158, 191 binary, 7, 9, 27, 84, 85, 143, 145, 243, 246, 249 birthplace, 191 bitmap, 83 bristles, 37, 41, 44 capacity, 83 combinatorial, 10, 35, 77, 79, 83, 115, 128, 143, 179, 212, 219, 233, 240, 246, 247, 251, 254, 259, 302 combinatorial vs. hierarchical, 78 decoding, 209 disc identity, 84, 85, 240, 251, 254, 302 Enigma, 33 general problem, xi, 37, 224, 255, 297 genetic, 255 histotypes, 149, 169, 213, 243, 246, 254, 262

445

Hox, 28, 191, 239, 240, 246–249 leg segments, 300 models, 7, 10, 212 Morse, 9 nonsense words, 7 Numb, 7, 9 patterns, 101 photoreceptors, 219, 224, 233 positions, 84, 101, 104, 124, 158, 213 transcription factors, 218, 219 transdetermination, 85, 169 zinc ﬁngers, 19 Cohen, Stephen, xii, 129, 137, 143, 157, 173 compartment boundaries, 4, 91, 105, 273. See also boundaries; cell lineage blastoderm A/P, 87, 89, 97 border zones, 107, 111, 125, 137–139, 142, 145, 150–159, 163, 173, 207 border zones, widths, 112, 137, 142–151, 167, 173, 287, 301 eye A/P, 91, 199, 201, 299 eye D/V, 165, 202–211 in growth control, 92, 118–119, 144, 148 kinks, 112, 148 lack of D/V in leg, 100, 111, 159, 170, 203 leg A/P, 61, 63, 89, 97, 100, 104, 105, 109, 114, 121, 125, 129, 201, 203, 289, 299 notum A/P, 193 peripodial membrane, 139 reestablishment of, 105, 123 role in disc initiation, 87, 91 thorax A/P, 89, 96, 97, 103, 111, 128, 148, 156, 287, 288 wing A/P, 87, 89, 92, 94, 105, 125, 137–154, 173, 177, 186, 189 wing D/V, 142, 146, 153, 159, 160, 173 compartments. See under cell lineage competence, 91, 227, 228, 235. See also under bristle patterns; cell signaling; prepattern based on transcription factors, 179, 217, 218 landscape, 84, 134 need for mitotic quiescence, 69, 304 region-speciﬁc, 109, 111, 117, 148, 153, 164, 166 states, 148, 217 suppression of, 135 vs. determination, 299 window, 28, 29, 72 computation. See also circuitry; computer metaphor absolute vs. relative, 47, 147, 167, 209 addition, 45, 72 arithmetic, 72, 73, 188 bristles, 71–74, 161 comparison, 47, 71, 72, 91, 167 critical mass, 133 distances, 173 division, 72 exponential, 72, 81, 83, 158, 301 Fibonacci series, 304 growth rates, 124 integration, 77, 124, 205, 288, 300 multiplication, 72 net force vectors, 137 patterns, xii ratio, 68, 72, 89, 124 shortest path, 93, 123 step function, 147 subtraction, 59, 61, 72, 128, 302

titration, 20–23, 71–73, 287 with RNA, 73 computer metaphor. See also circuitry; computation abstract symbolism, 9, 29, 72, 81, 89, 165, 243, 297 algorithms, 23, 31, 32, 72 binary digit (bit), 9, 84, 305 bitmaps and pixels, 83 cellular automata, 69, 235 cybernetics, 173 gating, 10 hardware vs. software, 255 inﬁnite loops, 137, 145 information processing, 84, 124, 205, 300 information theory, 81 input/output, 13, 17, 18, 23, 68, 72, 77, 123, 129, 131, 145, 163, 164, 209, 217, 246, 248, 254, 297–303 memory registers, 7, 10 modular subroutines, 7, 169, 299 program for building a bristle, 36, 297 program for building an eye, 255 program vs. blueprint, 297 resetting of variables, 7, 179, 182 servomechanisms, 153–155 Conway, John, 235 Cooper, Michael, 224 Couso, Juan Pablo, 300 Crick, Francis, 101 cuticle, xi, 5, 28, 87 pigmentation, 5, 28, 299 secretion, 5, 29, 40, 41, 73, 87, 199 thickness, 174 trichomes (hairs), 28, 65, 99, 159, 199, 247 two-dimensionality, 31 Dahmann, Christian, 151 Davidson, Eric, xiii, 244 Dearden, Peter, 303 Demerec, Milislav, 76 determinants asymmetric segregation, 7, 11, 24, 274 cytoplasmic, 9 heritable, 5 determination. See also cell states adult vs. larva, 9, 86, 87 all-or-none, 72 appendage tips, 72 binary decision trees, 27 bristle. See bristles endoderm, 72 gender. See genetics, sex determination glial cell fates, 7, 29, 72, 271 imaginal discs. See under imaginal discs leg vs. wing, 91 Malpighian tubule, 72 mesectoderm, 72, 177 muscle, 4, 9, 72, 259 neural. See neurogenesis potency, 91 role of HLH genes, 72 salivary ducts, 72, 91 stability, 86 states, 86, 90 states, maintenance, 90, 247–249 stepwise, 133, 302 tracheae, 72, 174 vs. competence, 299 vs. differentiation, 175, 185 wing veins, 177

446

development, stages of embryo. See under embryo larval instars, 49, 85, 87, 92, 121, 133, 148, 161, 208, 224 pupal period, 1, 92, 189, 208, 233 pupariation, xiii, 1, 85 Dexter, John, 298 Dietrich, Wilhelm, 202 differentiation, 5 bristle vs. sensillum, 28 bristle vs. vein, 175 general problem, xi macrochaete vs. microchaete, 29, 57 shaft vs. socket, 23 vs. determination, 175, 185 vs. growth, 196 waves, 208, 212, 215, 227, 229, 233, 305 DNA bending, 45, 259–265, 295 binding, 68, 72, 191, 219, 224, 248, 254. See also under protein domains, particular binding afﬁnities, 17, 45, 61, 84, 139, 259, 260, 261, 264, 275 binding screens, 61 binding sites, 15, 17, 19, 49–52, 61, 171, 172, 190, 248, 253, 259–265, 275, 281, 284, 287, 290, 295. See also DNA motifs binding sites, overlapping, 239, 295 binding sites, swapping, 171 binding speciﬁcity, 91, 259–263, 303 binding, competitive, 17, 18, 79, 84, 107, 182, 247, 278, 288, 291, 295 binding, cooperative, 79, 84, 263, 265 binding, nonspeciﬁc, 260 bookmarking, 249 chromatin, 248, 249 chromatin, spreading, 249, 281 cloning, 38, 40, 148 coding vs. non-coding, 41, 305 endoreplication, 29, 30, 41, 68, 86 euchromatin vs. heterochromatin, 43, 248 footprinting, 239 homology screens, 27 inverted repeats, 17 looping, 41, 45, 161, 191, 249, 261 methylation, 249 open vs. closed states, 249, 305 ploidy, 29, 86 replication, 249 DNA motifs. See also protein domains, particular E box, 17, 48–55, 68–72, 258, 263 homeobox, 28, 29, 85, 90, 115, 132–136, 149, 260–261 N box, 17, 50, 71, 258 T box, 112, 141 Dong, Si, 251 Driesch, Hans, 81, 93, 300, 302 Drosophila Aristotle’s “gnat”, xi, 256 artiﬁcial selection, 193 genus, 62, 63, 72, 135, 174, 237 giant, xi, 92 Hawaiian, xi, 67, 92, 193 hybrids, 71 melanogaster, xi, 202, 212, 298 other species, 31, 36, 63, 67 polymorphisms, 37 Dubinin, N. P., 37 Duncan, Ian, 251

INDEX

Ede, Donald, 224, 225, 305 embryo axes. See under axes blastoderm, 35, 77, 79, 89, 90, 97, 237 blastoderm cells, 77, 86, 87 blastoderm clones, 90, 199 cell transplantation, 77, 86 cleavage, 4 dorsal closure, 151 ectoderm, 77, 86–90, 109, 191 endoderm, 72 epidermis, 125 fate maps, 76, 77, 91, 245 gastrulation, 177 gliogenesis, 72 maternal gene products, 87 mesectoderm, 72, 177 mesoderm, 239 metameres, 76, 79, 84, 239, 246, 247, 254 myogenesis, 4, 9, 28, 72, 259 neuroectoderm, 50, 299 neurogenesis. See neurogenesis pair-rule stripes, 35, 45, 77–79, 84, 177 quirks of patterning, 86 salivary ducts, 72, 91 segmental gradients, 84 segmental identities, 79 segmentation, 76–80, 239, 299 segments vs. parasegments, 78, 84, 237–246, 305 stages, 85, 86, 90 tracheal system, 72, 151, 174 Wingless stripes, 78, 109 embryonic ﬁelds, 37, 43, 81, 86, 87, 190 emergent properties, 87, 89, 101, 107, 131–134, 142, 145, 153, 159, 160, 164, 177, 208–209, 213, 234, 236, 299, 302, 303 endocytosis, 26, 53, 180, 259 engineering, mechanical, 177, 227, 303 epidermis, 31 epiphanies, 270 equivalence groups, 37, 39, 43, 47, 49 evolution accidents, 31, 288 allometry, 299 arbitrariness, 158, 300 artiﬁcial selection, 31, 55, 193 Bateson’s Rule, 237, 300 by atavism, 246 by cobbling, xiv, 62, 297 by co-option, 10, 31, 72, 109, 136, 239, 246, 261 by crosslinking, xi, 36, 246, 255, 298, 305 by elaboration, 63 by gene duplication, 298 by gene sharing, 298 by genetic drift, 68, 71 by heterochrony, 69, 299 by heterotopy, 300 by hopeful monsters, 237, 246, 267 by tinkering, 63, 148, 255, 299, 302 by transposon jumping, 298 constraints, 92, 158, 300 default states, 299 entelechy, 300 epigenetic landscape, 300 evo-devo biology, 189 evolvability, 255, 264, 265 ﬁne-tuning, 228 frivolity, 31, 302 genetic workload, 247

improvisation, xiv inelegance, xiv, 35 legacies, 31, 132 loss through disuse, 68 missing links, 63 of anatomy, 255 of antennae vs. legs, 252, 300 of appendages, 243 of binding afﬁnities, 264 of body vs. leg segmentation, 136 of bract-bristle adhesion, 67 of bristle patterns, 31, 36, 62, 65, 69, 255 of bristles, 28, 29, 30 of circuitry, xi, 10, 31, 36, 63, 69, 72, 77, 136, 158, 173, 186, 223, 239, 244–247, 255, 262, 288, 299, 302, 305 of competence, 36, 246 of endocrine systems, 246 of eyes, 197, 223 of gene complexes, 36, 239, 243, 255, 298 of gene hierarchies, 303, 305 of genitalia, xii, 252 of halteres, 243, 246 of HLH genes, 68, 72, 305 of insects, 299 of leg segments, 300 of legs, 132 of metameres, 243 of modules, 63, 255, 264, 265, 297, 299 of opsins, 223 of organ shapes, 148 of protein domains, 257, 265 of proteins, 68 of receptor proteins, 286, 290, 297 of sex combs, 36, 63, 135 of sex determination, 72 of sexual behavior, 305 of tissue polarity, 235 of toggle switches, 255 of vein patterns, 174 of wings, 86 opportunism, xiv, 302 optimization, 79 reprograming, 10, 63, 72, 246 strategies, 165, 303, 305 vs. engineering, 158 eye anatomy, 197, 199 axes. See under axes ectopic, 252–254 equator, 198, 202–211, 296 equators, extra, 203, 207 fovea, 199 identity (“eyeness”), 240, 247, 302 missing, 169, 228, 231, 252 ommatidia. See ommatidia other insects, 212, 302 perimeter, 230 pigmentation, 197, 302 scar phenotype, 229 small, 169, 207, 228, 231 split phenotype, 17, 50 eye disc, 96 bipolar duality, 114, 169, 235, 302 cell death, 87 compartments, A vs. P, 91 fate map, 87, 199 gene expression patterns, 169, 203–209, 216–220, 233 head capsule, 204, 296 initiation, 77, 207

INDEX

invagination, 91 lattice tightening, 87, 227–228 maxillary palp, 191, 199, 240 MF (morphogenetic furrow), 44, 208–211, 215, 227, 233 MF engine, 229–234 MF initiation vs. progression, 231, 234–236, 253 MF vs. compartment boundaries, 229–234 MF, adhesive molecules, 281 MF, speed of, 229, 234 MF, straightening of, 151 MFs, collision of opposing, 236 MFs, extra, 212, 231–236 MFs, hot spot for, 231 mitotic band, 208, 211, 233 nomenclature, xiii, 199 ommatidia. See ommatidia peculiarities, 91 photoreceptors. See photoreceptors regional markers, 207 role of EGFR pathway, 179 role of peripodial membrane, 304 transcription factors, 182, 218 eye ﬁeld as a cellular automaton, 235 initiation, 253 margins, 231, 234–236, 253 MF initiation site, 235 mosquito, 212 fate maps, xiii, 92 embryo, 77, 91, 245 eye disc, 87, 199 leg disc, 96, 99, 105, 106, 111, 128 peripodial membrane, 87, 122, 139 wing disc, 94, 139, 156, 193, 301 ´ Fern´andez-Funez, Pedro, 160 Freeman, Matthew, 216, 217 French, Vernon, 93 Garc´ıa-Bellido, Antonio, 4, 32, 52, 85, 148, 155, 243 Gehring, Walter, xii gene complexes ANT-C, 237–243, 248, 260 AS-C, 17, 28, 30–75, 191, 193, 245, 272, 299, 305 Bar-C, 28, 134, 169, 194, 219, 220, 228, 233, 298 Brd-C, 272, 279 Broad-C, 20, 217, 223, 259 BX-C, 38, 45, 237–243, 246, 248, 260, 298, 299, 305 E(spl)-C, 10, 15, 17, 44, 48–51, 61, 68, 73, 218, 258 engrailed-invected, 141, 149, 243 homeobox, 243 homunculi, 45, 239 Hox, 239, 298 Iro-C, 96, 142, 166, 186, 187, 190–194, 203–208, 235, 236, 242, 243, 300, 302 Kni-C, 186 Spalt-C, 186, 187, 224 gene expression. See also under eye disc; leg disc; wing disc antenna, 91, 193, 205, 251, 288–296, 302 antenna vs. leg, 159, 251 arcs, 115, 127 at boundaries, 167, 171, 207, 209, 296

447

at interfaces, 87, 89, 107, 131–134, 142, 145, 153–155, 159, 160, 164, 177, 234, 302, 303 bands, 77, 127, 131, 136, 137, 141, 186, 205, 251 basal transcription level, 107 biscuits vs. bands, 151, 157 biscuits vs. doughnuts, 137, 138, 151 cages, 122, 123, 125, 137 cell-size independence, 92 circles, 104, 115, 117, 127, 129, 131, 137, 205, 251 coexpression, forced, 143, 157, 253, 265, 291 coexpression, natural, 59, 131, 135, 164–166, 171, 172, 179, 183, 208, 302 coexpression, paradoxical, 47, 91, 107, 114, 128, 139, 142, 219 coexpression, spatial but not temporal, 71 compartment-speciﬁc, 89, 104, 111, 112, 117, 121, 141, 146, 148, 153, 158–164, 203–207, 245, 285, 287 complementarity, 41–44, 59, 69, 78, 125, 129–135, 141, 143, 158, 169, 174, 175, 184, 188, 193, 301, 302 constitutive, 107 drivers, artiﬁcial, xiii, 7, 13, 30, 50, 51, 107, 113, 182, 216, 253 drivers, strong, 144, 157 drivers, weak, 138, 145, 154, 157 dynamics, 69, 78, 79, 127, 131, 134, 164, 167, 171, 188–191, 208, 218, 224, 234, 246, 247 enhancer traps, 112, 114, 127, 193, 205, 223, 300, 301 enzyme patterns, 301, 302 fuzzy zones, 128, 169, 193, 305 gradients, 127, 129, 135, 143, 177, 235 halos, 143, 156 hiatuses, 117 maintenance, 90, 107, 131, 160, 164, 166, 171, 247–249 nonfunctional, 172, 189, 218 nonuniform, 45, 69, 78, 84, 154, 243, 247, 249, 281, 305 on/off boundaries, 89, 121, 131, 136, 153, 157–160, 165, 173, 177, 186, 191, 203, 207, 209, 234, 235, 299, 303 on-then-off, 48, 91, 151, 184 overlaps, 77, 79, 128, 142, 145, 157, 167, 169, 172, 193, 251, 302, 303 overlaps, transient, 127, 129, 131, 134 parabolic regions, 104, 112 perdurance, xv, 125, 139, 147, 166, 167 periodic, 127, 136, 300 punctate, 157, 233 response to trauma, 98, 100, 119, 123 rings, 105, 112, 126–136, 141, 151, 205, 218 sectors, 104–106, 109, 112, 117, 121–128, 133, 141, 169, 205, 301 segmental vs. parasegmental, 246 single ﬁles of cells, 127, 135, 139, 145, 175, 177, 217 spreading, 119–122, 127, 145, 166, 171, 242, 288, 291 stratiﬁed (rainbow), 78, 131, 138–142, 157, 164, 188–193, 234 stripes, antenna, 251 stripes, antineural, 59–62, 69 stripes, ectopic, 68, 115, 117, 157, 171 stripes, embryo, 35, 77, 78, 89–91, 109, 293

stripes, eye D/V boundary, 207, 209 stripes, eye D-V axis, 233 stripes, eye furrow, 215, 233, 234, 304, 305 stripes, eye margins, 234 stripes, interveins, 177, 183 stripes, leg A/P boundary, 109, 111, 125, 129 stripes, leg disc, 112, 127, 128, 251 stripes, leg proximal-distal axis, 61, 112, 302, 304 stripes, notum, 104, 190–193, 304 stripes, pigment, 193 stripes, proneural, 59, 61, 62, 71, 163, 280 stripes, tarsus, 61, 302 stripes, widths of, 47, 59, 61, 89, 112, 127, 128, 137, 140–151, 163, 167, 173–177, 233, 287, 303 stripes, wing A/P boundary, 111, 128, 137, 142–147, 301 stripes, wing D/V boundary, 141, 146, 157, 159, 163, 164 stripes, wing disc, 141, 142, 157 stripes, wing veins, 47, 175, 177, 183–186, 303 gene families See also protein domains, particular Bearded, 17, 271, 272 Bright, 223, 292 Frizzled, 293 Gli, 287 GRIP, 290 Hedgehog, 105 Hox, 239, 248 IκB, 258 LIM-HD, 71, 260 MAGUK, 260, 283 Notch, 258 olfactory receptor, 191 POU-HD, 71, 260 Pox-Pax, 242, 262 Rel, 258 RTK, 179 Shc, 264 Smad, 290 steroid receptor, 217 Tcf, 258 TGF-α, 170 TGF-β, 105, 115, 189, 289, 290 Wnt, 105, 115, 292 gene groups Minute, 21, 29, 52, 56 Pc-G, 65, 111, 135, 207, 239, 242, 243, 247–251, 264, 300 Trx-G, 111, 239, 242, 243, 247–251, 276 gene regulation. See also circuitry activator-repressor switching, 19, 61, 107, 139, 255, 288 activators, 9, 13, 17, 51, 72, 73, 107, 133, 193, 239, 248, 249, 254, 261, 262, 290 activators, unknown, 107 auto-activation, 47–51, 72, 91, 107, 171, 193, 219, 233, 234, 248, 291, 295 auto-regulation, 72, 147, 171, 248 auto-repression, 17, 18, 107, 132, 147, 153, 166, 182, 225, 248, 263, 285, 288, 291, 295 basal transcription apparatus, 45, 298 batteries, 87, 217, 246 bipolar regulators, 19, 107, 255, 288 Britten-Davidson Model, 244 by cell-surface receptors (direct), 12, 175

448

gene regulation (contd.) by chromatin-remodeling, 17, 71, 111, 207, 239, 247–249, 264, 282, 295, 300 by micromanagers, 245–249, 253, 254 by nuclear import, 131, 133, 172, 181, 242, 251, 287, 288, 294 by proteolysis of regulators, 19, 20, 105, 107, 133, 182, 224, 263, 271, 295 cis-enhancers, 44–45, 79, 84, 92, 107, 128, 136 cis-enhancers, arrays of, 18, 21, 35–44, 52, 53, 72, 129, 131, 141, 164, 174, 188, 239, 289, 299 cis-enhancers, boundary vs. quadrant, 157, 164, 171, 245, 301 cis-enhancers, cell-type speciﬁc, 11, 48, 51, 219, 224, 254 cis-enhancers, colinear vs. scrambled, 41, 45, 239 cis-enhancers, constitutive, 171 cis-enhancers, disc-speciﬁc, 292 cis-enhancers, distant, 161 cis-enhancers, embryonic vs. imaginal, 245, 249 cis-enhancers, evolution of, 255 cis-enhancers, genomic repertoire of, 255 cis-enhancers, parasegment-speciﬁc, 243 cis-enhancers, region-speciﬁc, 28, 35, 36, 41–48, 191, 219, 239, 245, 249, 253, 289, 299 cis-enhancers, shared, 44, 298 cis-enhancers, stage-speciﬁc, 45, 86, 92, 128, 164, 174, 188, 227, 249, 289, 305 cis-regulatory region, 17, 71, 87, 239 cis-silencers, 62, 239 co-activators, 12–15, 91, 223, 264, 265, 288, 291, 295 combinatorial, 77, 136, 219, 245, 253, 254, 301 co-repressors, 12, 17, 49, 51, 61, 71, 207, 259, 264, 265, 281, 295, 302 default states, 173. See also under circuitry direct vs. indirect, xiii, 51, 107, 111, 128, 139, 141, 156, 164, 172, 187, 191, 193, 205, 219, 245, 248, 271, 288 enhanceosomes, 254, 255, 260, 300 enhancer-promoter bridging, 41, 45, 160, 161, 191, 279, 284 enhancer-promoter speciﬁcity, 45 hierarchies, 45, 69, 75–79, 83, 103, 133, 136, 141, 149, 182, 186, 217, 239, 244–248, 253, 303 histone acetylation, 248, 279, 288 histone deacetylation, 17, 248, 249, 275, 276, 281 insulators, 161 introns, 49, 171, 292 leg vs. wing disc, 137, 157, 164–170 licensing agents, 233, 254 locked-off vs. passive-off, 172, 173, 285, 294, 295 Mediator complex, 45, 135, 254, 300 networks, 79, 205, 246, 253, 303 open-for-business idea, 218 post-transcriptional, 48, 69, 72, 78, 79, 133, 234 post-translational, 68 promoters, 13, 15, 18, 45, 48, 50, 52, 55, 61, 72, 81, 253 pufﬁng cascade, 20, 217

INDEX

qualitative vs. quantitative, 44, 72, 81, 138, 139, 158 quenching vs. squelching, 17, 71, 79, 249, 295 Ras-dependent, 179 relays, 112, 141, 145, 177, 205, 215, 233 repressors, 13, 17, 18, 47, 48, 68, 72, 107, 135, 187, 190, 239, 248, 249, 261, 264 repressors, unknown, 171 stage-speciﬁc, 166, 246, 248, 249 synergy, 12, 52, 71, 75, 157, 181–185, 191, 252–254, 273, 275, 279, 281–283 trans-acting factors, 36, 43–45, 69, 72, 79, 245, 249, 298, 299 transcription cofactors, 190 transcription factors, 35, 51, 107, 181, 186, 190, 218, 260 transcription factors, landscape of, 301 triggers, 100, 133, 234 triggers, interface, 87, 89, 107, 131–134, 142, 145, 153–155, 159, 160, 164, 167, 171, 177, 207–209, 234, 296, 303 triggers, temporal, 91, 121, 123, 129, 137, 141, 145, 164, 299 genes. See also gene regulation; genes, particular; genetics; mutations; phenotypes; protein domains, particular abbreviations, xv, 37 adult vs. larval, 87 annulus, 127 antineural, xiii, 48, 53, 59, 62, 71–75, 163, 164, 180, 281, 282 autonomy in clones, 1, 33, 35, 39, 48, 50, 72, 80–85, 89, 124, 133, 141, 143, 148, 160, 165, 177, 183, 186–190, 209, 219, 245, 251, 282, 290 axis, 76, 147 boundary, 165 cascades, xiv, 20, 217 cassettes, xi, 47, 72, 305 cell cycle, 49, 258 cell type, 243 co-adapted, 71 co-expressed (naturally), 59, 135, 164–166, 171, 179, 208 colinearity, 45, 298 competence, 35, 45 cooperativity, 172 default states, 11, 59, 169, 171, 251, 299 disc-speciﬁc, xi, 159 dispensable, xiii, 24, 48, 86, 117, 157–159, 175, 182, 216, 218, 235 dispensable pairs, 79, 287 duplications, 36 early eye, 169, 235, 242, 252–254, 302 event-counting, 11 executive, 135 ﬁeld-speciﬁc, 242, 243, 254 gap, 77, 79, 84, 136, 147, 158, 186, 239 genomic repertoire of, 255 homeotic, 28, 65, 78, 83–86, 237–240, 247–249, 260 homologs, xii, 11, 13, 23, 26, 75, 109, 170, 179, 181, 239, 257, 274, 275, 283, 294, 304 housekeeping, 29, 85, 100, 302 Hox, 79, 80, 245–249, 254, 260 human, 13, 171, 254, 258, 259, 265, 290, 299, 303 instructive vs. permissive, xiv, 9, 36, 130–134, 189, 191, 215, 234, 249

interchangeability, 44 intronless, 17, 41, 73, 265 limiting factors, 21, 29, 69 mammals, 13, 15, 23, 26, 75, 225, 249, 258–265, 274, 275, 283, 295 map locations, xii master, 85, 171, 242, 252–254, 260, 297 maternal-effect, 76 memory, 10, 65, 81, 83, 84, 129, 158, 239, 248, 251 metamere identity, 239, 254 misexpression, xiii, 86 modiﬁers, 12 necessary vs. sufﬁcient, xiii, 37, 118, 132, 136, 245 nematodes, 261, 263, 295 neural precursor, 271 neurogenic, xiii nomenclature, xii nonautonomy, 39, 47, 48, 80, 133, 141, 177, 183, 186, 203, 209, 228, 231, 246, 303 on/off states, 1, 28, 79, 83, 84, 89, 107, 111, 143, 148–153, 158–160, 179, 239, 243–249 orthologs, xiii, 171, 258, 265, 283, 289, 290, 303 overexpression, xiii, 49 pair-rule, 18, 35, 77, 79, 84, 136, 158, 262, 278 pan-neural, 72, 182, 272, 274, 279, 284 paralogs, xii, 10, 141, 149, 181, 186, 219, 223, 253, 285, 289, 298, 303 pathways, xiii, xiv periodic-zone, 136 pinwheel, 97 plant, 239, 260, 261 prepattern, 27, 33, 35, 39, 45, 80, 148, 164, 193, 219, 278, 299 proneural, xiii, xiv, 37, 39, 41, 45, 49–53, 71–75, 161, 225, 229, 271, 279, 280, 283, 284 rate, 299 realizator, 134, 149, 174, 186, 246, 260, 305 redundant, xiii, 9, 10, 12, 17, 18, 21, 28, 40, 44, 45, 48, 55, 62, 75, 91, 125, 129, 134, 141, 149, 158, 167, 175, 218, 227, 231, 272, 278, 279, 281, 293, 295, 298, 300, 303 reporter, xiii, 28, 41, 48–52, 61, 117, 171, 191, 207, 219, 239, 291, 301 ribosomal RNA, 29 segmentation, hierarchy of, 76–80, 83, 103, 136, 186, 239, 247, 248, 303 segment-polarity, 79, 89, 97, 123, 131, 136, 158, 293 selector, 29, 48, 85, 89, 141, 145, 149–163, 173, 174, 188, 203, 246, 248, 262 selector, PI mode, 89, 145, 148, 163, 243 sensillar identity, 27, 75 sex determination, 30, 49, 72 subgenes, 37 switch, 9–11, 24, 28, 48, 85, 132, 148, 159–161, 165–169, 181, 184, 191, 207, 218, 240, 243, 255, 261, 271–284, 302 thin-zone vs. wide-zone, 136 ubiquitously expressed (naturally), 17, 49, 51, 59, 69, 75, 85, 92, 133, 158, 160, 161, 164, 225, 251, 263, 265, 274, 280, 282, 283, 290, 294 untranscribed, 191 upstream vs. downstream, xiv, 45

INDEX

vertebrate, xiii, 71, 109, 141, 179, 181, 225, 239, 258–261, 283, 285, 289, 290, 294, 305 work loads, 247 yeast, 258, 260, 261, 265 zygotic, 77 genes, particular 14-3-3ζ , 179, 180 18 wheeler, 300 abdominal-A (abd-A), 78, 239, 242 Abdominal-B (Abd-B), 78, 239, 242 abl oncogene, 223 abnormal chemosensory jump 6 (acj6), 271 abrupt, 303 Abruptex (=Notch), 52, 195, 279 absent solo-MD neurons and alfactory sensilla (amos), 27, 28, 75, 254, 276 absent, small, or homeotic discs 1 (ash1), 242 absent, small, or homeotic discs 2 (ash2), 242, 276 achaete (ac), 17, 30–75, 91, 112, 128, 141, 142, 161, 163, 167, 177, 187–195, 225, 229, 278, 279, 292, 296, 301–303 Actin5C, 138 Additional sex combs (Asx), 242 Antennapedia (Antp), 78, 81, 83, 85, 96, 131, 239, 242, 246, 249, 251 anterior open, 169 approximated, 185 apterous (ap), 127, 141, 158–173, 248, 260, 296, 301 araucan (ara), 142, 177, 185–194, 203–208, 235, 242, 289, 292, 296, 302, 303 arc, 292 argos, 52, 147, 170, 177–188, 194, 215– 218, 223, 227–229, 242, 303–305 aristaless (al), 92, 115, 117, 127–136, 141, 171, 251, 278, 292, 295, 300 aristapedia (=spineless), 80 Aristapedoid (Arp = Su(z)2), 242 armadillo (arm), 105–109, 118, 128, 134, 156, 163, 171, 173, 182, 194, 205, 242, 255, 258, 260, 294 arrow (arr), 106, 109, 205, 231, 293, 295 Arrowhead, 300 asense (ase), 17, 18, 37, 41, 48, 69, 272, 279 asteroid (ast), 185 atonal (ato), 27, 28, 73, 74, 212–235, 258, 276, 289, 296, 304, 305 baboon, 289 BarH1, 127, 131, 134–136, 193, 194, 205, 219, 220, 233, 276, 292, 296, 300 BarH2, 127, 134, 194, 219, 220, 233, 276, 300 bazooka, 11, 24, 271 Beadex, xiii, 71, 161, 260 Bearded (Brd), xi, 10, 12, 73, 272, 279 bicoid (bcd), 76–79, 84, 158, 239, 254 big brain (bib), 49, 55, 271, 279 Big brother, 223 bithorax (see Ubx), xiii, 80, 87, 237 blind spot (=poils aux pattes), 223 blistered (bs), 111, 141, 172–174, 177, 186–188, 245, 261, 289, 301, 303 bobbed, 29 Brachyury (vertebrate), 141 brahma (brm), 242 breathless, 179 bric a` brac (bab), 127, 129, 135, 251, 259, 300

449

bride of sevenless (boss), 15, 179, 180, 213–220, 224, 305 brinker (brk), 109, 112, 141, 142, 148, 157, 265, 291, 292 Brista (=Distal-less), 251 Broad-Complex, 20, 259 Brother, 223 Brother of Brd (Bob), 73 canoe (cno), 11, 75, 180, 263, 271, 279 Casein Kinase 2 (CK2), 109, 273, 293, 294 CASK, 260 castor, 217 caudal (cad), 77–79 caupolican (caup), 142, 177, 186–194, 203–208, 242, 289, 292, 296, 303 Cf1a (=ventral veinless), 260, 263 chaoptic, 223 chickadee, 29 Chip, 45, 160–166, 191, 260 Clock, 263 clown, 180 collier (=knot), 142, 259 comb gap (cg), 111, 135, 285, 292 congested-like trachea (colt), 174 connector enhancer of ksr (cnk), 180, 182 corkscrew (csw), 179, 180, 182, 303 costal2 (cos2), 109, 154, 157, 194, 286 cousin of atonal (cato), 272 cramped (crm), 135, 242 crooked legs (crol), 127 crossveinless, 189 crossveinless 2, 189, 289 crossveinless-like 6, 189 C-terminal Binding Protein (CtBP), 21, 61, 71, 190, 193, 259, 264, 273, 280, 282, 291 cubitus interruptus (ci), 105–112, 124, 128, 141–153, 165, 171, 186, 187, 205, 233, 242, 261, 285, 287 cut, 27, 141, 142, 161–166, 219, 220, 233, 242, 243, 259, 276 cycle, 263 Cyclin (genes A-F), 44, 49, 233, 260, 280 dacapo, 11 dachs, 135, 185 dachshund (dac), 127–135, 205, 220, 223, 228, 235, 242, 251–254, 261, 287, 292, 295, 300 dachsous, 185, 209, 293 dActivin, 289 dally-like (dlp), 293 dAP-2, 127 daughter of sevenless (dos), 179, 180, 182, 303 daughterless (da), 49, 50, 69–75, 113, 173, 174, 191, 223–228, 233, 263, 276, 280 Daughters against dpp (Dad), 109, 134, 141, 148, 167, 190, 290–295 dAxin, 109, 115, 118, 292, 294 DC0, 105, 109–119, 149–157, 187, 194, 231, 287, 303 dC3G, 180, 182 dCable (dCbl), 179–181 dCdc37, 179 dCdc42, 234 DCP-1, 228 dCul-1, 189 dE2F, 295 dead ringer, 223 deadpan (dpn), 17, 72, 127, 223, 265, 274, 300 dE-cadherin, 109, 153, 294

decapentaplegic (dpp), 39, 89, 90, 104–129, 133–134, 137–158, 170–177, 185–194, 205, 228–235, 245, 252, 253, 288, 289, 295, 301, 303, 305 Deformed (Dfd), 78, 239 Delta (Dl), 9, 12, 15, 25, 47–50, 59, 61, 71, 75, 124, 127, 135, 160–169, 173–177, 203–211, 215, 216, 223–229, 242, 254, 259, 271, 272, 280, 296–300, 303–305 deltex (dx), 12, 15, 135, 175, 272, 280 Dense, 278 derailed, 179 Dfrizzled2 (Dfz2), 105, 109, 125, 146, 158, 190, 194, 293, 295 Dfrizzled3 (Dfz3), 104, 112, 125, 141, 142, 158, 295 dHSF, 254 Dichaete, 31, 45, 194, 195, 265, 280 disconnected (disco), 127, 136 discs large, 271 discs overgrown, 263 dishevelled (dsh), 15, 106, 109, 114–118, 124, 131–136, 163, 167, 193, 194, 205–209, 242, 279, 293 dispatched, 125, 185, 285 Distal-less (Dll), 29, 86–90, 96, 104, 115, 117, 127–137, 141, 142, 148, 156, 158, 205, 242, 247–254, 292, 295, 300–303 division abnormally delayed (dally), 105, 109, 146, 194, 290, 293 dJun, 179–182, 209, 215, 220, 293 dLim1, 127, 135, 240 dMyc, 92 Domina (=jumeaux), 278 dorsal, 254, 255 Dorsal switch protein 1 (Dsp1), 242 Dorsal wing (Dlw), 159–163, 166, 167 doublesex, 30 double-time (=discs overgrown), 263 downstream of receptor kinases (drk), 179–182, 303 dPax2, 28, 29, 219, 220, 233 dRac1, 180, 182, 293, 295 dRacGap, 180, 182 dRaf, 107, 153, 169, 170, 179–188, 194, 231, 242, 258 dRasGap, 304 Dredd, 228 drICE, 228 drifter (=ventral veinless), 174 Drop, 228 Drosophila CREB-binding protein (dCBP), 107, 109, 111, 288, 291, 295 Drosophila Dr1-associated protein (dDrap), 71 Drosophila Heat shock protein 90 (dHsp90), 180 Drosophila MAPK-ERK Kinase (dMEK), 169, 179–182, 304 Drosophila Mitogen-Activated Protein Kinase (dMAPK), 169, 179–183, 216, 218, 223, 227, 235, 259, 304 dSara, 289 Dscam, 191, 261 dShc, 179, 180, 182, 303 dTrk, 179 dumpy, xi, 174, 259 DWnt-4, 292 ebi, 180 ebony, 193 ecdysoneless, 278

450

genes, particular (contd.) echinoid, 223 echinus, 278 embryonic lethal, abnormal vision (elav), 223, 273 enabled, 300 engrailed (en), 61, 78, 79, 87–91, 107, 111, 112, 117, 121, 124, 137–163, 170, 182, 187–189, 205, 209, 224, 229, 239, 246, 248, 288, 299, 301, 303 Enhancer of Ellipse 24D (=echinoid), 180 Enhancer of Ellipse, 24D (=echinoid), 230 Enhancer of split (=m8), 9, 17, 272, 281 Enhancer of zeste, 135 Epidermal growth factor receptor homolog (Egfr), 29, 56, 73, 106, 153, 169, 170, 173, 177–188, 194, 205, 212, 215–218, 227, 228, 242, 264, 281, 288, 297, 300, 303, 305 Epithelial Adenomatous Polyposis Coli (E-APC), 109, 258, 294 escargot (esg), 89, 91, 127, 131–136 even skipped (eve), 45, 78, 262 expanded, 209, 289, 293 extra eye (ee), 242 extra sex combs (esc), 80, 135, 242, 249, 265 extradenticle (exd), 111, 131–134, 166, 195, 205, 242, 251–254, 260–262, 300–302 extramacrochaetae (emc), 47, 48, 71–75, 141, 142, 161, 174, 175, 193, 194, 223, 233, 234, 260, 281, 301–303 eye gone (eyg), 223, 235, 242, 252, 262 eyeless (ey), 80, 205, 228, 235, 237, 242, 249, 252–254, 292, 296 eyelid (=osa), 223, 292, 305 eyes absent (eya), 205, 220, 228, 235, 242, 252–254, 261, 287, 292 fasciclin II (fas II), 127, 131, 134, 151, 281 fat facets (faf ), 180, 223 fat-head, 107, 153 ﬂamingo (=starry night), 209, 293, 304 forked, 29 four jointed (fj), 127, 135, 185, 205, 301 fringe (fng), 15, 52, 135, 159–167, 173, 194, 203–208, 242, 279, 300 fringe connection (frc), 292 frizzled (fz), 125, 131, 136, 146, 158, 181, 205, 209–211, 293, 304 fruitless, 305 fused (fu), 109, 149, 185, 286 fushi tarazu (ftz), 18, 77, 78, 239, 254 futsch, 215 gammy legs (gam), 106, 292, 295 Gap1, 179–182 Geranylgeranyl transferase-1 (GGT1), 179, 180 giant (gt), 78 glass, 219, 220, 233, 254, 289, 305 glass bottom boat (gbb), 143, 185, 189, 194, 289, 290 glial cells missing, 271 grain, 112, 300 grainyhead, 217 grim, 228 gritz, 170, 180, 184, 218 groucho (gro), 17, 49–52, 61, 71, 106–111, 135, 153, 155, 164, 173, 175, 223, 142, 254, 255, 265, 281, 291, 295, 298, 305 gurken, 170 gustB, 271 Gαi, 271 H15, 112, 114, 133, 296

INDEX

hairless (H), 18, 20–28, 51, 52, 75, 175, 273, 282, 305 hairy (h), 17, 32, 45–49, 61–63, 68–75, 112, 127, 128, 136, 163, 177, 187, 193, 195, 205, 223, 233, 234, 264, 265, 282, 289, 292, 296, 302, 304, 305 head involution defective (hid), 228 heartless, 173, 179 hedgehog (hh), 79, 89, 90, 104–112, 117, 119–129, 137–160, 165, 171, 174, 177, 185–187, 194, 205, 208, 212, 223, 228–235, 242, 253, 285, 288, 292, 301, 305 homothorax (hth), 112, 127–136, 141, 148, 166, 205, 242, 251, 249–254, 261, 262, 292, 296, 300–302, 305 huckebein, 265 hunchback (hb), 78, 79, 217, 239, 254 hyperplastic discs, 135 inscuteable (insc), 24, 271 Insulin receptor, 179 invected (inv), 141, 145, 149, 153, 161, 301 I-POU (=acj6), 71, 260 irregular chiasm C-roughest, 215 jumeaux, 271 Jun N-terminal Kinase (JNK), 180, 209, 259, 293 kakapo, 257 karst, 180 kekkon1 (kek1), 177–183, 303 kinase suppressor of ras (ksr), 179, 180, 182, 258 kismet (kis), 242 klingon, 218 klumpfuss (klu), 48, 127, 271, 282, 300 knirps (kni), 78, 141, 174, 177, 186, 239, 264 knirps-related, 186 knot, 139, 142, 147, 177, 183–187, 259, 289, 301, 303 kohtalo, 223 Kruppel (Kr), 78, 239, 254, 264 ¨ kuzbanian (kuz), 49, 53, 135, 175, 258, 282 l(1)ts504, 135 l(3)1215, 300 L38, 257 labial (lab), 239, 242 leg arista wing complex (lawc), 242 lethal (2) giant discs (l(2)gd), 115, 157 lethal (2) giant larvae (lgl), 24, 273 lethal at scute (l’sc), 17, 30, 37, 41, 45, 50, 68, 69, 73, 271 lilliputian (lilli), 180, 289 lines, 295 liquid facets (lqf ), 180, 223 Lobe, 207, 292 lozenge (lz), 219, 220, 223, 233, 276 m8 and other E(spl)-C genes, 15, 17, 50, 61, 73, 127, 142, 164–169, 175, 177, 205–209, 219–223, 233, 264, 265, 271, 272, 278, 281, 300, 301, 303, 305 many abnormal discs, 290 master of thickveins (mtv), 142, 148, 189, 288, 301 mastermind (mam), 169, 175, 273, 282 maverick, 289 Medea, 109, 290 Merlin, 289 Minute (multiple genes), 21, 29, 52, 56, 201 miranda, 24, 271 mirror (mirr), 186, 190–194, 203–208, 234, 236, 242, 296 misshapen, 209, 293

moira, 107, 242 Mothers against dpp (Mad), 109, 126, 128, 131, 141, 143, 147, 171, 190–191, 229–235, 253, 255, 290 multi sex combs (mxc), 135, 242 multiple ankyrin repeats single KH domain (mask), 180 musashi (msi), 24, 264, 273 muscleblind, 220 myoglianin, 289 naked cuticle, 292 naked cuticle (nkd), 112, 134, 167, 230, 295 nanos, 76, 79 nemo, 292, 303 net, 173, 174, 177, 185, 303 neuralized (neu), 147, 156, 273, 282 NK-4, 255 Notch (N), xiv, 9, 12–17, 25, 47, 49, 61, 71–75, 106, 114, 124, 135, 147, 157, 160–177, 185, 188, 194, 203–211, 215–219, 227, 229, 235, 242, 245, 259, 264, 271, 273, 278, 283, 297–299, 301, 304, 305 Notchless (Nle), 283 Notum, 242, 292 nubbin (nub), 127, 167, 171, 263, 301 numb, 5–27, 72, 165, 259, 264, 266, 270, 274, 286 numb-associated kinase (nak), 24, 274 Numblike (mouse), 15 odd Oz (odz), 127, 136, 278 odd paired (opa), 262 odd skipped (odd), 78, 79, 127, 134, 136 Odorant receptor 83b (Or83b), 191 onecut, 220 ophthalmoptera (opht), 242 Ophthalmoptera (Opt), 203, 242 optix, 235, 242, 252, 253 optomotor-blind (omb), 111–113, 118, 125, 133, 140–143, 154, 155, 158, 187, 205, 245, 291, 292, 296, 301, 303 oroshigane, 185, 285 orthodenticle, 205, 220, 289, 296, 300, 305 osa, 242 paired (prd), 262, 264 paired box-neuro (poxn), 27, 127, 262, 277 pangolin (pan), 105, 106, 109, 128, 171, 173, 190–194, 255, 260, 295 pannier (pnr), 41, 45, 151, 190–194, 234, 235, 242, 292, 295, 302 partner of inscuteable, 271 partner of numb, 24, 264, 271 patched (ptc), 109, 111, 124, 125, 138–157, 177, 185–187, 194, 208, 231, 242, 285, 288, 301, 303 pdm-1 (=nubbin), 11, 217, 297 pdm-2, 11, 297 period, 262, 263, 302 Phospholipase Cγ , 179, 181, 182, 223, 303, 304 phyllopod (phyl), 20, 182, 219, 220, 224, 271 PI3 kinase, 303 pipsqueak, 220, 300 pleiohomeotic (pho), 127, 135, 242 plexus, 173, 174 poils aux pattes, 135 pointed (pnt), 153, 169, 179, 181, 182, 194, 219, 220, 242 polychaetoid (pyd), 30, 71–75, 263, 283 Polycomb (Pc), 107, 135, 148, 160, 242, 248

INDEX

polycombeotic (=Enhancer of zeste), 242 Polycomblike (Pcl), 65, 242 polyhomeotic, 107, 111, 122, 128, 149, 155, 288 porcupine (porc), 106, 242, 293 postbithorax (see Ubx), 87 Posterior sex combs (Psc), 242, 271 Presenilin (Psn), 53, 283, 295 prickle (pk), 131, 136, 209, 293, 300 proboscipedia (pb), 135, 239, 242 prospero (pros), 11, 24, 182, 219, 220, 224, 233, 254, 274 Protein Kinase A. ( See DC0) Protein Kinase C (PKC), 109, 263, 294 Protein Phosphatase 2A. ( See twins) Protein Tyrosine Phosphatase-ERK/Enhancer of Ras1 (PTP-ER), 179, 181, 304 puckered, 278 punt, 106, 109, 114, 115, 118, 129, 145, 185, 188, 190, 194, 229, 235, 289 radius incompletus, 177 Rap1, 179–181 Ras GTPase-activating protein (RasGAP), 181 Ras1, 29, 153, 169, 179–182, 194, 223, 228, 242, 254, 304 rasputin (rin), 181 reaper, 228 reduplicated, 123 retina aberrant in pattern (rap), 212, 223 RhoA, 209, 293 Rhodopsin (genes #1-#6), 197, 223, 224, 253, 254 rhomboid (rho), 141, 173–189, 194, 217, 223, 227, 303 roadkill, 289 rolled (rl=dMAPK), 173, 177, 180, 183, 185, 233 rotund (rn), 127 rotundRacGAP (=rotund), 29, 181, 182, 189 rough, 213, 215, 218–224, 228, 233, 234, 289, 305 Roughened (=Rap1), 181 rudimentary, 29 rugose, 223 runt, 264 sanpodo (spdo), 11, 29, 271 saxophone (sax), 143, 189, 289–291 scabrous (sca), 15, 30, 50, 55, 62, 75, 147, 157, 165, 215, 219, 223, 225–227, 233, 283, 289, 305 scalloped (sd), 142, 164, 171–173, 242, 265, 271, 296, 301, 302 schnurri (shn), 109, 188, 291, 292 scratch, 223, 274 screw, 289 Scruffy, 141 scute (sc), xiv, 17, 20, 30, 31, 37–75, 91, 113, 142, 161, 163, 191, 193, 223, 225, 245, 283, 292, 296, 301, 303 Scutoid, 278 semang, 181, 182, 223 senseless (sens), 50–52, 72, 220, 233, 271, 284, 302 Serrate (Ser), 15, 24, 25, 52, 55, 127, 135, 158–169, 173, 175, 203–209, 225, 242, 274, 296, 300, 301 seven in absentia (sina), 20, 182, 219, 220, 224, 271

451

sevenless (sev), 179, 180, 213–220, 227, 302, 303 seven-up (svp), 181, 215–224, 233 sex comb distal, 135 Sex comb extra (Sce), 242 Sex combs on midleg (Scm), 242 Sex combs reduced (Scr), 80, 112, 239, 242–248 shaggy (sgg), 109–118, 130, 132, 156, 157, 163, 190, 194, 205, 278, 294 shattered, 223 shaven (=dPax2), 28 shibire (shi), 26, 49, 52, 53, 59, 146, 175, 275, 284 shifted, 285 short gastrulation (sog), 185, 188, 303 shortsighted, 289, 305 shotgun (shg = dE-cadherin), 109 sine oculis (so), 205, 220, 228, 235, 242, 252–254, 261, 287, 292 singed (sn), 29, 39 single-minded (sim), 177, 262 skinhead, 169, 242, 292 sloppy paired, 84 smoothened (smo), 105, 109, 111, 128, 149–154, 185, 208, 229, 231, 286 snail (sna), 91, 264, 278 Son of sevenless, 179, 181, 182, 304 spalt, 30, 125, 140–143, 154, 155, 158, 174, 177, 185–187, 194, 205, 220, 224, 233, 245, 251, 252, 291, 292, 296, 301–303 spalt-related (salr), 141, 185, 186, 245, 301 spineless (ss), 29, 91, 127, 135, 205, 242, 249–254, 300 spiny legs (sple=prickle), 67, 131, 293 spitz (spi), 48, 52, 89, 91, 147, 169, 170, 174, 179–185, 194, 215–218, 227, 228, 242, 303, 304 split (see Notch), 15, 17, 50, 272, 281 split ends (spen), 181, 292 sprint, 181 sprouty (spry), 174, 179–185 Src42A, 181 Star, 29, 177–185, 217, 228, 303 starry night, 209, 293, 304 strabismus (=Van Gogh), 209, 293 strawberry notch, 135, 166 string, 11, 30, 44, 92, 223, 233, 271, 296, 302 Stubble, 29 sugarless (sgl), 106, 290, 293 sulfateless (sﬂ), 293 super sex combs (sxc), 242 supernumerary limb (slimb), 105, 109, 111, 115, 149, 154, 157, 164, 180, 242, 287, 294 Suppressor 2 of zeste, 271 Suppressor of deltex (Su(dx)), 272, 278 suppressor of forked (su(f )), 100, 119, 123, 135, 242 Suppressor of fused (Su(fu)), 109, 154, 286 Suppressor of Hairless (Su(H)), 9–25, 49–52, 135, 158, 164, 169, 171–175, 209, 219, 224, 255, 275, 284, 297 suppressor of Hairy wing (su(Hw)), 161 TACE, 258 TAF250, 254 tailless (tll), 223, 239 tam (=polychaetoid), 281, 283 tango (tgo), 29, 161, 242, 252, 263 target of poxn (tap), 258, 271 tartan (trn), 127 tartaruga (trt), 292

teashirt (tsh), 127, 134, 136, 166, 207, 242, 252, 295 tetraltera (tet), 242 thick veins (tkv), 106, 109, 114–118, 124, 125, 129–134, 141–148, 177, 185–189, 194, 229–235, 288–292, 301, 303 timeless, 263 tolkin, 185, 189 Toll-like receptor, 300, 301, 305 tolloid, 189 torso, 179, 304 tout-velu, 185 tramtrack (ttk), 11, 18, 19, 169, 182, 218, 220, 224, 242, 255, 259, 271, 275 tricornered, 29 trithorax (trx), 107, 242, 248 Tubulinaα1, 138, 154 Tufted, 278 tumorous head 1, 278 twin of eyegone (toe), 235 twin of eyeless (toy), 205, 235, 242, 253, 254 Twin of m4 (Tom), 73 twins, 11, 24, 109, 157, 179, 181, 275, 289, 294, 295 twist, 239, 254 two-faced (tfd), 242 UbcD1, 182 Ultrabithorax (Ubx), 78, 80, 87, 112, 237, 239, 242–253, 299, 305 ultraspiracle, 217, 220 u-shaped (ush), 45, 190–194, 292, 302 Van Gogh, 209, 293 vein (vn), 141, 153, 167–186, 205, 216, 227, 288, 289, 296, 302, 303, 305 ventral veinless (vvl), 141, 142, 171, 174, 177, 189, 263, 296, 303 vestigial (vg), 86, 89, 91, 139–142, 156–158, 164–173, 242, 245, 265, 292, 296, 301, 303 VP16 (herpes virus), 13, 51, 61, 254, 284 vrille, 289 white (w), 201 wingless (wg), 15, 30, 55, 78, 79, 87–91, 104–129, 133–142, 146, 156–173, 189–194, 205, 230–236, 242, 245, 279, 289, 292, 295, 301, 302 yan, 179, 180–182, 194, 220, 259 yellow (y), 1, 17, 36, 39, 41, 43, 45, 67, 193, 201 genetics. See also mutations; phenotypes analysis, xiv, 62, 202 artifacts, xiv, xv background effects, 36, 38, 45, 69 chimeric transgenes, 13, 18, 51, 68, 126, 147, 209, 216, 224, 284, 303 complementation, 37 constructs, constitutively active, xiii, 12, 51, 114, 124, 141, 145, 151, 170, 173, 190, 207, 216, 234 constructs, dominant-negative, 169, 195, 216, 242 constructs, inversion recombination, 43 cosuppression, 298 deletion analysis, 41 developmental vs. physiological, 299 dosage compensation, 71, 248 dosage effects, 13, 21, 23, 29, 30, 31, 49, 52, 68, 69, 71–75, 113, 175, 208–211, 243, 246, 287, 297 dosage screens, 68 enhancer maps, 41, 239 enhancer traps, xiii, 165, 219, 282, 296, 301

452

genetics (contd.) epistasis, xiii, xiv, 48, 109, 160, 161, 175, 179, 183, 186, 188, 209, 271, 278, 287 epistasis, paradoxical, xiv ﬂp recombination, xiii ﬂp-out trick, xiii, 185 Gal4-UAS method, xiii genome project, xi gynandromorphs, 1–4, 36, 39, 52, 67, 77, 86, 91 haplo-insufﬁciency, 21, 29, 50, 52, 113, 160, 175 heat-shock promoters, xiii, 7, 69 history, xv, 12–15, 32, 37–43, 57 inhibition by RNAi, 271 intersexuality, 63, 72, 246 inversions, 298 LOF vs. GOF testing, deﬁnitions, xiii molecular, 167 mosaic analysis, 9, 23, 33, 35, 39, 45, 52, 56, 69, 80, 85, 104, 114, 128, 151–155, 209, 213, 218, 302 mosaic analysis vs. Gal4-UAS method, 118 nomenclature, xiii nonspeciﬁc effects, 249 penetrance vs. expressivity, 159 phenotypic rescue, 13, 18, 21, 47, 51, 71, 118, 151, 154, 158, 160, 164, 165, 173, 182, 186, 187, 188, 207, 216, 218, 223, 228, 231, 233, 235, 253, 274, 281, 283, 290, 291 pleiotropy, 302 ploidy chimeras, 55, 56 ploidy effects, 55, 56, 62 saturation of limiting factors, xiii, 12, 69 sex determination, 1, 30, 36, 49, 63, 72 somatic recombination, 33 techniques, xiii temperature-dependence, 39 transpositions, 255, 298 transvection, 159 ubiquitous overexpression, 28, 45, 48, 68, 69, 75, 96, 109–119, 141, 145, 147, 182–186, 207, 215, 218, 233, 235, 246, 247, 276, 282 unequal crossing over, 134, 298 variegation, 3, 4, 248 vs. genomics, 254, 256 Gerhart, John, 255 Ghysen, Alain, xii Gibson, Matt, 122 Gierer, Alfred, 184 Goldschmidt, Richard, 39, 73, 237, 299 G´omez-Skarmeta, Jos´e Luis, 41, 44 gradients back-to-back, 89, 148, 209, 211 biphasic (spire-ramp), 158 bootstrapping, 153–55 cell-size independence, 92 centrifugal, 129 cone-shaped, 93, 94, 101, 103, 104, 127, 129, 131, 227 contour lines, 31, 94, 103, 128, 139 contour maps, 100 curved, 101, 104, 124, 126, 128 developmental capacity, 93 double, reciprocal, 78, 124, 147, 291, 304 embryo, 76, 84, 109, 113, 158 eye disc, 205, 209, 211, 215 heterochronic, 158, 209 leg disc, 111, 124, 129

INDEX

leg segments, 80, 135 linear vs. exponential, 81, 83, 158, 301 merging, 156 mirror-symmetric, 89, 97, 145, 148, 163, 167, 209, 211 notum, 104, 190–193 nuclear import, 133 of gene expression, 127, 129, 135, 143, 177, 235 overlapping, 78, 101, 104, 124, 291, 304 parabolic, 128 parasegmental, 84, 89 peaks vs. valleys, 156 perception vs. reality, 125, 134, 143, 147, 158, 188–190, 291 proneural, 39, 163, 164 saddle-shaped (composite), 134 sawtooth, 80 seamless appearance, 153–156 segmental, 83, 84 shaping, 158 sliding, 156 slopes, 81, 92–94, 125, 147, 158, 205, 227, 291, 301 slopes, opposing, 78, 124, 147, 291, 304 smoothing, 147, 158 tent-shaped, 101, 167 theory, 81, 83, 92, 93, 124, 158, 209, 299 wing disc, 111, 138–146, 156–158, 186, 301 wing veins, 185, 188 Greenwood, Simon, 231, 234 growth advantage, 201 anti-apoptosis factors, 118 asymmetric, 154 blastemic, 96, 123 cell-density independence, 94 cessation, 92, 94, 99, 122, 124, 155 compensatory, 89, 91 control, 86, 92, 124, 155 control by patterning system, 124, 134, 148 disc, 57, 85, 89, 92 disproportionate, 91, 123, 150, 155, 156, 252 distal, 100, 101, 103, 115, 133, 171 even vs. uneven, 33, 35, 92, 148, 158 extended period of, 92, 121, 155 factors, 92, 98, 115, 118–119, 124, 196, 283, 293 goal of, 91 gradients, 63 hyperplastic, 63, 80, 85, 92, 115–119, 124, 135, 143, 147, 157, 185 intercalary, 67, 93–95, 103, 123, 124, 133, 153–156 intrinsic limits, 92, 155 larval, 86 maintenance, 92, 122, 148 mitogens, 98, 118–119, 143 need for Dpp, 92, 118–119, 144, 148, 155 need for various pathways, 92 orthogonal, 118 potential, 93, 106, 124 rate, 4, 85, 91, 105, 133, 148, 150, 151, 153, 202, 305 region-speciﬁc, 87, 92, 133, 134, 153 role of nitric oxide, 92 stunting, 158, 164 timing, 1 vs. differentiation, 196 vs. patterning, 141, 158

vs. transdetermination, 85, 169, 249 wound-induced, 96, 98, 122 Gubb, David, 97 Hadorn, Ernst, 299 Haerry, Theodor, 143 Halder, Georg, 237, 252 Hanson, Thomas, 202 Hartenstein, Volker, 77 Heberlein, Ulrike, 229 Heitzler, Pascal, 52 histoblast nests, 1, 2, 73, 87, 299 history, xv bristle pattern research, 32, 57 debate about AS-C, 37–43 debate about eye compartments, 202–203 debate about Fz-Dfz2 redundancy, 293 debate about Notch, 12–15 debate about SOP mitoses, 5 debate about sternopleura, 86 embryo segmentation, 76 embryology, 173 epochs and eras, 57, 86, 97–99, 105, 119, 202, 254 GDC Model, 92–96 genetics, 202, 254, 256 genetics, molecular, xii, xv, 97 ironies, 4, 33, 79, 103, 139, 156, 167, 246, 252, 256, 287 Morgan’s team, xii, xiv, 1, 32, 33, 37, 86, 123, 298, 299 PC Model, 93–105, 113, 122–124, 154, 156 PC vs. Boundary Model, 103–105 prepattern vs. positional information, 79–84, 251 signal transduction pathways, 105 studies of discs, xii, 76 utility of studying, 254 histotypes, 9, 72, 81, 85, 169, 189, 242, 243, 254 Holtfreter, Johannes, 304 homeosis, 84, 123, 160, 237–255. See also genes: master; mutations: homeotic amnesia, 249 antenna-to-leg, 80–85, 96, 129, 249–252, 299 context-dependence, 252 correlated with regeneration, 85, 246 correlated with tissue loss, 85 deﬁnition of, 80, 237 etiology, 85 eye-to-antenna, 169 general problem, 81, 237 haltere-to-wing, 80, 87, 243, 246 inter-compartment, 87, 148, 149, 158–161 inter-disc, 84, 85, 171 inter-leg, 65, 80, 87, 243 inter-segmental, 243 intra-disc, 169 leg-to-wing, 115 notum-to-wing, 169, 302 of head, 141 paradoxical, 65 patchy, 81, 83 single-cell. See cell states: switching vs. transdetermination, 85, 169, 237, 242, 249, 254, 302 wing-to-haltere, 151, 243 wing-to-notum, 169, 302

INDEX

homology antenna-leg, 81, 83, 249–252, 299 bristle-scale, 67 eye-wing, 203, 205 inter-leg, 63, 65, 89 shaft-socket, 23 veins-bristle rows, 187 wing-haltere, 89, 249 Huang, Franc¸oise, 57 imaginal discs. See also imaginal discs, particular adepithelial cells, 87 basal lamina, 87 cell cycle. See mitosis cell death. See apoptosis; cell death co-culturing, 118 compartments. See cell lineage; compartment boundaries conjoined, 98, 100, 123, 156 culture, 84, 92, 94, 96, 169, 175, 203, 212, 248 cytonemes, 67, 125 deﬁnition of, 1 delamination, 87 determination vs. differentiation, 86, 91 diploidy, 86 disc-speciﬁc markers, 86, 89, 91, 171 dissociation-reaggregation, 65, 85, 90 duplication vs. regeneration, 92–97, 100, 103, 121–124, 139, 156, 169 duplications, 86, 99, 115, 157 embryonic origin, 1, 77, 85–91, 171 epidermal cells, 86 epidermal vesicles, 132, 149, 151, 153, 190 epithelial folds, 2, 87, 124, 139, 245 epithelial folds, extra, 115, 135 epithelial puckering, 151 epithelium, 46, 65, 69, 87, 197 epithelium, columnar, 87, 121, 122, 139, 233 epithelium, pseudostratiﬁed, 87 evagination, 1, 57, 65, 87, 89, 139, 174, 199 extracellular matrix, 124, 174 extracellular space, 124 ﬁlopodia, 53, 65, 67, 87, 125, 174 fusion, 123, 150, 151, 156 geometry, 99, 121 growth. See growth histology, 4, 86 history of studies, xii, 76 hollow sacs, 1, 87, 122 hyperplasia, 85, 115, 117, 119, 135, 157, 185 identities, 86, 90, 149, 249, 254 identities of compartments, 85, 89, 107, 145, 148, 150, 153, 158–163, 173, 188, 203, 248 identities of subﬁelds, 171, 240, 302 identity codes, 84, 85, 240, 251, 254, 302 insularity, 87 invagination, 85, 87, 91, 99 irradiation, 86, 95, 97, 99, 100, 106, 135, 139 maceration, 96 mesoderm cells, 85–87 mitosis, resumption of, 85 mitotic arrest, 85 mitotic orientations, 87, 133 mitotic rates, 85, 87, 153, 299 myoblasts, 87

453

necrosis, 113 neurons, 87 numbers of cells, 85, 86, 169 peripodial membrane, 87, 93, 97, 121–123, 139, 205, 304 planarity, 87 prematurely metamorphosed, 133 regeneration, 86, 92–96, 100, 103, 299 regeneration, polarity of, 94, 95, 100, 103, 304 regeneration, types of, 123 regenerative potential, 93–96, 100–105, 123, 139 rotation, 89, 199, 205, 207, 299 size, 67, 85, 92, 245 stalk, 87, 89, 99, 199, 234 surgical fragmentation, 92, 94, 95, 99, 100, 103, 121, 139, 169 tissue removal, 95–100, 103 tracheae, 86, 87, 139, 174 trans-lumenal extensions, 87 trans-lumenal interactions, 96, 122, 123, 139, 212 transplantation, 92, 94, 169, 175, 203, 212, 248 tumors, 85 wound healing, 85, 96, 100, 103, 121, 122, 156 imaginal discs, particular, 2 clypeolabral, 2, 77, 86, 199 eye. See eye disc genital, xii, 1, 2, 4, 77, 87, 91, 123 haltere, xii, 4, 77, 85, 86, 171, 239, 243, 245, 299 humeral, 2, 77, 87 labial, 2, 4, 77, 86, 169, 199, 247 leg. See leg disc wing. See wing disc Ingham, Philip, 79 insects, xi, 67, 72 cockroach, 93, 98, 119, 135, 300 Drosophila. See Drosophila epidermis, 87 grasshopper, 49, 129, 151, 300 hemimetabola, 105, 133, 135 hemiptera, 49, 59, 224 holometabolous, xi honey bee, 223 hymenoptera, 76, 223 lepidoptera, 29, 31, 47, 61, 65, 67, 132, 299 mosquito, 212 other dipterans, 67, 69, 76, 86, 99, 193, 255 paleoptera, 243 vs. humans, 197 vs. mammals, 13, 179, 297, 302 vs. nematodes, xii, xiii, 1, 11, 50, 247 vs. plants, 262 vs. slime molds, 105, 151 vs. vertebrates, xi, xiii, 86, 93, 151, 197, 199, 224, 261, 297, 304, 305 wasp, 302 Internet databases, xii, xiii, 112, 205, 257, 301 Jan, Lily Yeh, 10 Jan, Yuh Nung, 10 Karch, Franc¸ois, 305 Karlsson, Jane, 105 Kauffman, Stuart, 84, 249 Kirschner, Marc, 255

larva, xi Bolwig’s organ, 285 brain, 92 denticle belts, 79, 285 development, 86, 87 dimensions, 2 discless, 86 epidermis, 77, 86, 87, 99 eye organ, 289 fat body, 92 growth, 86 Keilin’s organ, 99 midgut, 77, 289, 291 neurons, 99 polyteny, 86 Lawrence, Peter, 32, 84, 89, 101, 148 Lebovitz, Richard, 212 Lecuit, Thomas, 129, 141, 143 leg anatomy, 99, 127, 131 axes. See under axes branched, 99–103, 115, 123 claws, 4, 75, 99, 103, 106, 115, 117, 118, 127, 251, 252, 299 clone stripes, 133 cockroach, 93, 98, 119, 135, 300 deformed, 106 evolution, 132, 300 feathery, xi femur-tibia detachment, 135 fused 1st legs, 97 fused segments, 80, 128, 135 grasshopper, 129, 151, 300 hemimetabolous insects, 105, 133, 135 identity (“legness”), 240, 249, 254 identity (vs. antenna), 83, 246, 249–254 intersegmental membranes, 80, 300 joints, 135, 136, 205, 299, 300 joints, extra, 131, 135, 136 pretarsus, 134, 151 regeneration, 133, 135 segmental gradients, 80, 83, 135, 300 segmental identities, 129–135, 300 segmentation, 80, 128, 131, 135–136, 300 segmentation, leg vs. body, 136 short, 65 trochanter boundary, 132 truncated, 118, 119, 129, 132 leg disc antineural stripes, 128 bipolar duality, 105, 106, 114 border cells, 135 compartments, 4, 63 dispensable regions, 119 distalization, 115–119, 129–134 Dpp (vs. Wg) as a major mitogen, 118–119 Dpp and Wg as morphogens, 111–113 Dpp-Wg circuitry transition thresholds, 117 Dpp-Wg cooperation in distalization, 115–118 Dpp-Wg mutual antagonism, 113–115 duplications, 115, 132 endknob, 87, 100, 103, 114, 117, 131 extra, 115 fate map, 96, 99, 105, 106, 111, 128 folds, 87, 133, 134, 136 folds, extra, 115, 135 gene expression patterns, 89, 109, 111–117, 121, 126, 127, 131–135, 243, 245, 251

454

leg disc (contd.) geometry, 99, 105, 121, 128, 129, 137, 301 growth control center, 119 initiation, 77, 89, 91, 114, 117, 129, 132 mitoses, region-speciﬁc, 133 modes of morphogen transport, 124–125 outgrowths, 100, 115–119, 124, 132 outgrowths, converging vs. diverging, 119 proximal-distal axis, 115–118, 129–135 proximal-distal zonation, 127, 131–135 proximal-vs.-distal cell afﬁnities, 132 quirks, 123, 132, 133 regional markers, 113, 114, 115, 117, 129, 134 role of adepithelial cells, 87 role of Dll, 129–132 role of Hh-Dpp-Wg circuit, 105–119, 128–129 routes of morphogen transport, 125–128 spot, Dll-sensitive, 129 spot, peripodial Hh, 121–123 spot, quiescent, 92, 117, 135 sternopleura, 86, 89, 99, 106, 118, 127, 299 tip speciﬁcation, 115–118 topology (vs. body segment), 109 upper medial quadrant, 97, 99, 101, 122, 123, 156 vs. wing disc, 137, 170, 171 Lewis, Edward, 243, 244, 246, 298 ligands. See also gradients; morphogens afﬁnity for receptor, 52, 124, 125, 165, 293 anti-apoptosis, 118 cell-surface, 53, 135, 167 cis interactions, 52, 61, 73, 211 cleavage from precursor, 48, 53, 126, 146, 181, 182, 217, 259, 285, 289 cognate receptors, 9 diffusible, 1, 46, 49, 53, 61, 89–91, 111, 121, 142, 145, 146, 150, 153, 169, 180, 181, 211, 215, 217, 225, 285, 289, 292 diffusion range, 55, 89–92, 107, 111, 113, 117, 119, 124–125, 140, 145–158, 183–188, 215, 218, 227, 285–288, 304 diffusion range, modulation of, 125, 126 diffusion rate, 125, 143, 147, 183, 193, 215, 285, 286 effective range, 125 endocytosis, 27, 59, 124 epitopes, 126 exocytosis, 124 extinction, 50 gradients, 147 homodimerization, 289 ingestion, 175, 179, 213, 215 membrane-bound, 53, 179, 272, 275, 280 proteolysis, 158 redundant, 24 regulation by receptor, 166 regulation of receptor, 125, 158, 166, 288, 292, 295 release, 125 short-range inducers vs. long-range morphogens, 101, 104, 107, 109, 115, 117, 138, 187, 234, 304 tethering to lipid rafts, 125 transcytosis, 27, 124 unknown, 29, 53, 119, 127, 131, 133, 134, 223, 227, 293 Lindsley, Dan, xii links. See also circuitry deﬁnition of, xiii

INDEX

fallacious, 143–148 for bipolar duality, 114, 169, 302 for cell memory, 248 for computing SOPs, 48 for creating boundaries, 107, 111, 145, 149 for disc identity, 239, 243–252 for disc initiation, 89–90 for ensuring single SOPs, 47, 50–52 for explaining regeneration, 121–123 for eye’s A-P axis, 229–235 for eye’s D-V axis, 205–211 for making bristle patterns, 59, 75, 190–194, 278–284 for making bristles, 11–20, 25, 271–275 for making eyes, 205 for making gradients, 142 for making leg segments, 300 for making legs, 113–119, 128–135 for making ommatidia, 219–227, 305 for making sensilla, 27, 276–277 for making wing veins, 175, 186–187, 303 for making wings, 158, 165–171, 301 for rewiring other links, 253–255, 302 for signal transduction, 109, 125, 137–148, 158, 181, 285–296 for wing’s A-P axis, 145, 156 for wing’s D-V axis, 156, 161–164 inconsequential, 158 wiring, cis-trans, 136, 246 logic. See also circuitry Boolean, xiii, 17, 25, 79, 113, 114, 117, 128, 130, 134, 157, 171, 172, 193, 213, 215, 219, 251, 252, 254, 289, 292, 295, 296, 299, 300 disc development, 256 double-negative, xiii, 47, 51, 78, 107, 187 grammar, 20, 254, 300 jigsaw-puzzle, 163, 219, 252, 254, 261, 300 nucleosome, 239, 305 of chemical reactions, 100 promoter, 17, 18, 122, 254, 298 subtractive, 59, 61, 128, 141, 163, 302 syntax, xiii, 10, 18, 79 Venn, 79, 112, 117, 133, 158, 166, 171–173, 251, 302 Ma, Chaoyong, 229 Maynard Smith, John, 32 mechanisms, 270. See also circuitry actomyosin motors, 26 bootstrapping, 153, 155 cell alignment, 151 cell shaping, 23, 44 clocks, intrinsic vs. extrinsic, 11, 215, 217 clocks, oscillator, 297 counting, 10, 11, 73, 297, 304 dominoes, 11, 215 error-correction, 73, 148 gating, 10, 11, 21, 69, 233, 287 inert decoy, 50, 68, 71, 72, 160–163, 180, 216, 260 lateral inhibition, xiv, 39, 47, 49, 55, 59, 175, 184, 215, 224–227, 304 licensing, 11, 44, 233, 254 nucleosome displacement, 248 quality control, 73 ratcheting, 10, 11, 81, 121, 143, 147, 155, 217, 243, 244 reaction-diffusion, 33, 69, 184, 225 schedule, 11, 215 servomechanisms, 153–155

shufﬂing of zinc ﬁngers, 20 stripe-adding, 78, 87, 107, 131, 133, 134, 142, 145, 153–155, 159, 160, 164, 177, 208–209, 234, 302, 303 stripe-splitting, 59, 61, 62, 78, 79, 128, 142, 163, 166 time window, 10, 68, 71–74, 216, 246, 247, 299 timing, 11, 53 vesicle transport, 27, 124, 265 weird, 87 Meinhardt, Hans, 100, 101, 184 Mentzel, Christian, 298 Merriam, John, 4 metamorphosis, xi, 76, 86, 87 metaplasia, 84 Micchelli, Craig, 173 microbeam irradiation, 76 microcautery, 76, 95, 97, 106 Mil´an, Marco, 173 mitosis arrest, 24, 44, 69, 85, 92, 117, 135, 151, 160, 234, 304 asymmetric, 7, 11, 24 asynchronous, 9 cell cycle, 11, 24, 44, 49, 85, 100, 223, 233, 234, 265, 304 licensing, 44, 233 nuclear shifts, 304 orientation, 3, 4, 7, 24, 87, 133 quiescent spots, 44, 69, 92, 117, 135 quiescent zones, 92, 151, 160, 233, 234, 304 radiosensitivity, 41 random vs. patterned, 92, 208, 233 rate, 4, 81, 85, 87, 91, 151, 153, 202, 299 regulators, 92 spindle, 3, 24 synchrony, 44, 85, 87, 208, 211, 215, 233, 234, 305 syncytial, 79, 86 timing, 174 zonation in wing, 175 Mlodzik, Marek, 225, 231 models, 266–268 active-cell vs. passive-cell, 125 Bateson’s toy, 99 cellular automata, 235 computer, 79, 224, 236, 303 hybrid, 84, 101, 103, 158 intransigence, 147 mathematical, 79 overturned, 13, 38, 39, 44, 48, 84, 87, 123, 134, 145, 147, 158, 185, 202, 211, 224, 247, 253 resurrected, 13, 79, 115, 156, 224, 299 robustness, 99 utility, xiv, 32, 123 Modolell, Juan, 37, 57 Mollereau, Bertrand, 224 Morata, Gines, 89, 148 Morcillo, Patrick, 161 Morgan, Thomas Hunt, xi, 31, 193, 256, 298, 302 morphogenesis, xi, 23, 87, 124, 131, 134, 135, 148, 174, 304 morphogens. See also gradients; ligands; morphogens, particular; positional information active transport, 101, 124, 125 as mitogens, 92, 118–119, 148 as survival factors, 92, 118, 228

INDEX

binding by lipids, 124 binding by proteoglycans, 124 channeling, 101, 124, 126 circumferential, 101, 104 constraints on usage, 158, 249 constructs, membrane-tethered, 139, 145, 153, 157, 185, 285, 293 deﬁnition of, 81, 138 diffusion range. See under ligands dorsalizing vs. ventralizing, 104, 109, 111–113 mistaken expression, 85 modes of movement, 104, 124–125, 146 passive diffusion, 124, 126, 130 polarized movement, 125 pumping, 129 rate of transport, 125 response modulation, 84, 125, 134, 158 retrograde transport, 125 role of actin, 125 role of cell shape, 124 role of dynamin, 125 role of receptor density, 125, 158, 188 spheres of inﬂuence, 106, 111, 124 tagging with GFP, 147 transcytosis, 27, 124, 146 transport via cytonemes, 125, 146 transport via lipid rafts, 125, 146 morphogens, particular Bicoid, 77, 83 Decapentaplegic, 89–91, 104–129, 133–137, 140–148, 153–158, 171, 177, 185–196, 229–234, 254 Hedgehog, 84, 89, 90, 104–112, 117–129, 137–146, 150, 153, 177, 185–187, 196, 229–234, 254 Spitz, 89, 91, 196, 215, 254 tip (unknown), 83, 100, 103, 104, 122, 129, 251 Wingless, 84, 89, 90, 104–129, 133–137, 142, 146, 156–158, 161, 171, 189–190, 196, 234, 254 Wnt (unknown), 113, 132, 133, 136, 205, 231, 292, 293, 304 Muller, H. J., 32, 37, 39 mutations. See also phenotypes affecting bristle patterns, 194, 278–284 affecting bristles, 271–275 affecting bristles and sensilla, 276–277 allelic series, 129 allelic speciﬁcity, 20, 24, 37, 38, 39, 44, 45, 132, 284, 291 antimorphs, xiii atavistic, 36, 63, 67, 135, 174, 243, 246 breakpoints, 41, 43, 44, 129, 194, 195, 280 cell-lethal, 63, 97, 98, 100 deletions, 18, 23, 28, 35, 41, 43, 44, 48, 50, 51, 59, 69, 71, 134, 164, 185, 186 dominant vs. recessive, xii, 129 dominant-negative, 173, 234 duplications, 23 heterochronic, 299 homeotic, 63, 65, 80, 83, 135, 169, 237, 240, 243, 246–249, 261, 300, 302 hypomorphs, 7, 23, 37, 59, 73 idiosyncratic, 52, 75, 87, 158, 288 inversions, 41, 43, 44 lethal, 87, 141 lethal phase, 86 lethal side effects, 80 macromutations, 237

455

maternal effect, 278 missense, 15 Morgan’s quest for, 37 neomorphs, xiii, 18, 87, 149, 271, 278, 284, 288, 302 nomenclature, xii, 37 nonsense, 44 null vs. leaky, xii, 10, 21, 23, 75, 118, 138, 154, 157, 183 null, compound, 37, 44, 69, 107, 114–119, 125, 132, 149, 153–157, 165, 188, 207, 215, 216, 218, 227, 231, 273, 275, 282, 291, 303 null, utility of, 43, 44, 117, 118, 293 P-element insertions, 49 point, 41, 44, 298 prepattern, Stern’s quest for, 80, 252 rearrangements, 186 screens, 5, 18, 26, 37, 87, 141, 160, 305 screens, dosage, 68 screens, modiﬁer, 12, 180 temperature-sensitive, xiii, 15, 23, 26, 59, 63, 73, 121–123, 138, 139, 156, 169, 173, 182, 229, 235, 283 X-ray induced, 37 mysteries, 268–270 Needham, Joseph, 301 Nellen, Denise, 141, 143 nervous system antigens, 208, 213, 215 axon projections, 55, 97, 191–196 cell lineage, 4, 11 embryo, 49, 50, 52, 73, 149 gene expression, 27 genetics, 271, 278 grasshopper, 49 imaginal discs, 87 lobes, antennal, 191 lobes, optic, 141, 196, 198, 285, 292 midline fates, 262 mutation screens, 5 neurons, 27, 173 repressors, 20 wiring, 5, 20, 27, 55, 99, 139, 191–196, 199, 228 Neumann, Carl, 157 neurogenesis adult, 31 axon pathﬁnding, 20, 55, 99, 174, 191–96 axons, retrograde transport, 125 bristle. See bristles embryo, 24, 31, 166 ganglion mother cells, 11, 24 HLH genes, 72 neural competence, 49 neuroblasts, 7, 9, 11, 24, 49, 51, 52, 77, 191, 196, 217, 274, 297 N¨othiger, Rolf, xii ¨ Nusslein-Volhard, Christiane, 76 Oberlander, Herbert, xii Occam’s Razor, 35 O’Farrell, Patrick, 124 O’Keefe, David, 173 ommatidia bristles, 30, 57, 199, 209, 218 cell types, 197, 199, 208–211, 215, 223, 227, 228, 233 chirality, 209–211 cone cells, 208, 212, 215–219, 228

ﬁring center (at equator), 305 founder cell (R8), 213–218, 227, 233 lattice, 32, 201, 202, 208, 224, 227, 230 mystery cells, 208, 211, 223 number per eye, 197 photoreceptors. See photoreceptors pigment cells, 209, 212, 216, 218, 228, 229 polarity, 81, 198, 207, 203–211, 236, 293 rate of creation, 208, 227 rhabdomere trapezoids, 197, 209, 211 role of Delta-Notch, 53, 211 rotation, 131, 201, 208–211, 304 spacing, 212, 304 stages of development, 208, 211, 215, 217, 225, 227, 233 Pan, Duojia, 252 pattern formation. See also axes; boundaries; circuitry; morphogens; prepattern; positional information; rules alignment, 67, 148–153 amphibian limbs, 93 arthropods vs. vertebrates, 93 Bateson’s Rule, 99–101 by balkanization, 131, 148, 190, 301, 302 by chain reactions, 111, 156, 167, 215, 235, 297 by determination waves, 211, 212, 235, 305 by drawing lines, 97, 173, 179 by elaboration, 63, 133, 302 by intersecting lines, 35, 45, 89, 90, 101, 157, 234 by invisible scaffolds, 156 by iteration, 29, 74, 135, 145, 217, 300 by painting sectors, 97 by painting stripes, 61–62, 69, 78 by physical forces, 33, 35, 135, 175, 189, 237 by sequential induction, 139, 211, 215 by signal relay, 139, 186 by slicing a cylinder, 136 by stratiﬁcation, 87, 107, 131–134, 142, 145, 153–155, 159, 160, 164, 177, 208–211, 234, 302, 303 by symmetric annealing, 136, 167, 174, 177 by tessellation, 224 by triangulation, 56, 59, 224, 305 cell death, 87 cell lineage, irrelevance of, 3 cell-size independence, 92 checkerboard, 50, 158 community effect, 85 continuity, maintenance of, 99, 101 control of growth, 124, 134, 148 David vs. Goliath power, 123 development vs. regeneration, 94, 97, 123 discontinuities, 93, 95, 105, 124, 154–156 discs vs. embryo, 97 disruption, 80 distalization, 100, 115–119, 129–134, 157, 158, 166, 171 duplication vs. regeneration, 92–97, 100, 103, 121–124, 139, 156, 169 duplications, 86, 99, 105, 115, 154 duplications, mirror-image, 89, 94–99, 103–106, 113, 118, 122, 128, 129, 148–165, 169–173, 275 during growth, 86, 94, 133 edge effects, 93–96, 103, 153, 305 epimorphosis vs. morphallaxis, 96, 103, 123

456

pattern formation (contd.) evolution, 86 feather lattice, 224, 305 ﬁngerprints, 151, 199, 299 general problem, xi generic tools, 179 homeogenetic induction, 85 hypertrophy. See growth: hyperplastic in a syncytium, 86 intercalation, 93–96, 133, 153–156 local vs. global, 21, 33, 50, 55, 56, 62, 68, 79, 81, 92, 94, 128, 139, 173, 196, 211, 212, 224, 235, 236, 254, 301 molecular basis, 79 noise, 201, 202, 228, 229 number control, 136, 304 organizers, 100, 129, 301 out of phase, 84, 225, 227, 233 precision, 31, 46, 62, 69, 86, 99, 151, 156, 201, 228, 305 principles, 270 quirks, 86, 299, 305 reconstitution, 67, 90, 156 regeneration, 81, 86, 93–95, 100, 103, 123, 156, 246, 299, 304 repatterning, 67 reprograming, 63, 246 robustness, 91 scaffolds, 79, 80, 148 short-range inducers, 101, 104, 107, 109, 115, 117, 138, 187, 234, 304 subpatterns, 20, 62, 154, 156 top-down vs. bottom-up, 56 topographic cues, 69 trans-boundary cooperation, 189 triplications, 98, 99, 100, 119, 254 vertebrate somites, 304 vs. growth, 141, 158 without positional information, 133 patterns. See also anatomy; bristle patterns; phenotypes geometry, 31, 32, 56, 59, 93, 123, 160, 189, 201, 211, 212, 304 geometry, leg vs. wing, 99, 105, 128, 137, 139, 301 hoops, 135, 151 ladders, 90, 189 lattice, 32, 201, 202, 224, 227, 230, 305 mammal coat color, 302 orthogonal, 137, 189 pigmentation, 32, 43, 75, 193 Peifer, Mark, 305 phenotypes Bateson’s sports, 99 bizarre, xii, 1, 30, 53, 63, 94, 98, 115, 117, 119, 124, 143, 145, 153–155, 157, 203, 237, 248, 252, 254, 265 converging vs. diverging outgrowths, 98, 100, 101, 103, 119 deﬁcient (but not duplicated), 106, 118, 139, 169, 170 discless, 87 disc-speciﬁc, 87 duplication-deﬁcient, 97, 106, 113, 139, 155, 169 etiologies, 80, 96, 119, 123, 128, 131, 136, 153, 154, 157, 163, 249, 262, 271 hyperplasia, 63, 80, 115–119, 124, 135, 143, 147, 157, 185 Janus, 106, 113, 118, 128, 129, 153, 159, 163, 169

INDEX

leakiness, 21 mirror-image, 63, 89, 95–100, 105, 106, 113, 118, 122, 128, 129, 148–165, 169–173, 275 paradoxical similarity of LOF and GOF, 160, 163, 171, 286 phenocopies, 80, 99, 185, 189, 237 quantitative spectrum, 156 segment-polarity, 136 sensitive periods, 26, 63, 98, 123, 135, 170, 175, 182, 189, 216, 229, 237, 252, 284 syndromes, 49, 80, 99, 131, 136, 149, 164, 165, 182 triplications, 98, 99, 100, 119, 254 photoreceptors anatomy, 197, 199 axon projections, 196, 198 developmental sequence, 208–215, 218, 227, 233 gene expression, 220 identity code, 219, 224, 233 inner vs. outer, 197, 199, 233 markers, cell-type speciﬁc, 215, 218, 220 nuclear shifts, 304 R1-R6, extra or missing, 216, 218, 220, 224 R3-vs.-R4 determination, 209–211 R7 equivalence group, 182, 215 R7, extra or missing, 180, 213–218, 220, 224 R8 equivalence group, 225 R8 spacing, 55, 62, 225 R8, extra or missing, 223 R8, need for atonal, 28, 72, 74, 217, 220 R8, similarity to SOP, 225 rhodopsin subtypes, 197, 223, 224, 253, 254 vs. bristles, patterning of, 57, 225 Plunkett, Charles, 39 Poodry, Clifton, xii, 39 positional information. See also axes; boundaries; gradients; morphogens abdominal segments, 84, 141 absurd aspects, 158 area codes, 81, 85, 191 azimuths, 106, 124 clockface metaphor, 103, 155 coordinate systems, 56, 79, 81, 83, 96, 103, 105, 193 coordinate systems, bipolar, 106 coordinate systems, Cartesian, 90, 93, 101, 105, 157–158 coordinate systems, polar, 93, 95, 97, 101, 105, 124, 126, 128 coordinate systems, tripolar, 101, 106 coordinate systems, warped, 122 coordinates, polar, 103 crowded coordinates, 99, 103, 105, 122 deﬁnition of, 81, 84 down-the-slope constraint, 93 emission vs. reception, 148, 151 French Flag metaphor, 81, 84, 139, 299 graininess, 83, 92, 158, 299 individuation, 84, 89, 145, 148, 163 interpretation, 81, 83, 84, 209, 251 interpretation modes, 83, 84, 89, 167, 249, 251, 300 interpretation, minor role of, 158, 301 intrasegmental, 83, 135 PI-prepattern hybrid, 84, 158 positional values, 81, 83, 84, 93, 96, 99, 248, 251

positional values, misleading idea of, 158, 301 precision, 81, 124, 158 recording, 81, 83, 84 reference lines, 87, 89, 95, 109, 151, 188 reference points, 55, 59, 90, 101, 157, 234 singularities, 93 speciﬁcation, 81, 83, 89, 251 speciﬁed vs. determined states, 90, 111 stages, 81 thresholds, 84, 104, 109, 113–117, 126–131, 138–147, 154–158, 183–193. See also under circuitry; prepattern thresholds, setting, 167 thresholds, sharpening, 291 universality, 81, 83, 84, 96, 202 vs. prepattern, 83, 79–84 wiring of genome, 41, 83, 84 zonation, 78, 84, 107, 125, 127, 131, 138–143, 163, 188–193, 252, 301 zonation, orthogonal, 142, 158 Posthlethwait, John, xii prepattern, xiv, 33, 43, 68, 301 absurd aspects, 80, 83 antenna vs. leg, 83, 251 competence, 35, 39, 47, 83 deﬁnition of, 33 embryo segmentation, 79, 84 evolution, 36 factors, 45, 47, 69, 73, 75, 190, 193, 229, 305 genes. See under genes gradients, 56 hypothesis, 33, 35, 39, 79–83, 251 landscape, 62, 69, 193 macrochaetes, 35, 68, 190–193 mutants, 80, 252 nodes, 33, 79, 80 overlapping, 83 pair-rule stripes, 78, 79 PI-prepattern hybrid, 84, 158 proneural clusters, 55, 68 proneural vs. antineural, 164, 195 reaction-diffusion, 33, 83 saga, 80 sex comb, 80, 135 singularities, 33, 35, 36, 80, 83, 84, 158, 301 singularities, cis-enhancers for, 35, 92 singularities, cryptic, 36, 41, 43, 68, 135 thresholds, 83. See also under circuitry; positional information universality, implausible, 80–83 vindication, 79, 190, 251, 299 vs. positional information, 79–84 programming. See under computer metaphor protein domains. See also protein domains, particular alterations, 72, 300 dimerization, 15, 17, 161, 216, 248, 280, 283 docking, 68, 158, 300 domain-swapping experiments, 179, 260 idiosyncratic, 28 interchangeability, 257 masking vs. unmasking, 15, 21, 261, 263, 273, 274 overlapping, 15, 21 propellers, 249, 265 reference sources, 257 repeats, deﬁnition of, 257 repeats, extreme number, 259

INDEX

scaffolding, 257, 263, 293 self-defeating, 288 size range, 257 steric effects, 72 protein domains, particular, 257–265 14–3–3, 179, 258 activator vs. repressor. See under gene regulation (activators or repressors) ADAM, 258 ankyrin, 12, 13, 180, 258 arm, 258 basic, 17, 23, 161 bHLH, 17, 28, 29, 41, 48, 61, 74, 91, 177, 217, 225, 239, 242, 249, 258, 259, 272 BTB, 18–20, 135, 259 bZip, 180 COE, 186, 259 cut, 259 DEF, 180, 181, 259 DEJL, 181, 259 DNA-binding, 17–23, 28, 61, 68, 71, 85, 107, 141, 148, 161, 163, 171–174, 190, 218, 223, 239, 242, 257–265, 275, 277, 282, 288–291, 295, 303 EF hand, 295 EGF-like, 15, 52, 165, 170, 180, 181, 259 EH, 26, 259 Ets, 181, 182, 259 F, 180, 260 glycosylation sites, 15, 289, 292 GUK, 260 helix-turn-helix, 260, 263, 291 HLH, 39, 49, 50, 68, 71–73, 161, 260, 297 HMG, 180, 242, 260, 295 homeo, 11, 24, 107, 129, 135, 158, 161, 186, 190, 191, 203, 213, 217, 223, 235, 239, 242, 249–252, 260–263 Hox hexapeptide, 254, 260, 261 Ig-like, 170, 180, 181, 261, 263 KH, 180 kinase, 179 leucine zipper, 261 LID, 161 LIM, 135, 136, 158, 161, 261 LNG, 261 MADS, 174, 186, 239, 261, 290 NES, 262 NLS, 12, 13, 15, 20, 180, 262 opa, 141, 180, 262 Orange, 61, 71 paired, 252, 262 PAS, 29, 177, 249, 262 PDZ, 180, 263, 292 PEST, 12, 263 PH, 180, 181, 263 POU, 167, 217, 263, 297 PRD, 264 PTB, 24, 180, 264 PxDLSx(K/H), 71, 264 RAM23, 13 RNA-Binding, 181, 264 runt, 264 SH2, 180, 181, 264 SH3, 180, 181, 264 signal, 265 Sox, 45, 265 sterol-sensing, 125, 285 TALE, 254 TEA, 171, 265 transmembrane, 13, 15, 261, 285 VP16, 13, 51, 61, 284

457

WD, 24, 249, 265 WD40, 180 WRPW, 17, 51, 61, 71, 255, 265 zinc ﬁnger, 18–20, 48, 50, 52, 91, 107, 127, 132, 134, 136, 141, 180, 186, 190, 217, 223, 224, 239, 242, 252, 265 proteins. See also genes, particular; proteins, common; proteins, modiﬁcation of adaptors, 24, 109, 179, 180, 254, 263, 264, 294, 304 adhesive, 134, 150, 151, 153, 173, 174, 177, 261, 299 binding afﬁnities, 259, 264, 265, 291, 294, 295 bipolar, 19, 107, 255, 288 bridging, 41, 45, 160, 161, 191, 279, 284, 287, 294 caspases, 228, 262 catalysts, 13 cellomics, 298 chimeric, 13, 18, 51, 68, 126, 147, 209, 216, 224, 284, 288, 303 chromatin-remodeling, 17, 71, 111, 207, 239, 247–249, 264, 277, 282, 295, 300 competitive binding, 17, 18, 68, 72, 79, 84, 161, 170, 215, 278, 291, 295 conformational changes in, 26, 258, 261, 264, 286, 290 constant shape vs. variable sequence, 260, 263, 264 convertases, 53 cooperativity, xiv, 79, 84, 261, 263, 265, 290, 300 cytoskeletal, 180 cytoskeletal regulators, 181 deacetylases, 293 dehydrogenases, 293 dimerization, 17, 49, 79 DNA-binding. See under protein domains geranyl transferases, 180 GFP-tagged, 126 glypicans, 290, 293 GTP exchange agents, 180, 181 GTPases, 26, 180, 181, 275, 293 GTP-binding, 26 heterodimers, 12, 15, 17, 21, 49, 50, 68, 71–75, 133, 171, 172, 190, 193, 251, 252, 258–264, 272, 276–280, 284, 290, 303 homodimers, 15, 17, 71, 72, 146, 181, 258–263, 272, 273, 280, 286, 289 huge vs. tiny, xi, 174, 180, 259 immunological detection, 13, 27, 41, 43, 53, 126, 147, 303 immunological tracking, 175 isoelectric point (pI), high, 21 isoforms, 18, 19, 181, 182, 222–224, 239, 247, 258, 273–275, 281–284, 287, 294 isoforms, extreme variety of, 191, 261 kinase inhibitors, 11 kinases, 24, 180, 213, 263, 286, 287, 293, 294 ligands. See ligands lipases, 181, 304 metabolic enzymes, 29, 85, 302 mimicry, 68 multi-protein, 254 multi-protein complexes, xiv, 11, 41, 182, 191, 224, 247, 248, 252, 261, 265, 271, 279, 280–289, 294–296 multi-protein machines, 300 neuronal, 191

nomenclature, xii nuclear import, 9–11, 15, 20, 21, 131, 133, 171, 172, 251, 258, 286–295 oligmers, 258 oligomers, 181, 259, 262, 273, 275, 290 phosphatases, 11, 24, 157, 180, 181, 275, 289, 294, 304 polyadenylases, 100 proteases, 15, 53, 180, 189, 228, 258, 265, 289 proteoglycans, 55 proteolysis, 19, 20, 105, 133, 180, 182, 218, 224, 263, 271, 280, 294. See also under receptors proteolysis, proteasome-dependent, 12, 107, 111, 262, 263, 271, 287, 288 receptors. See receptors RNA-binding, 24, 181, 264 saturation, 9 scaffolding, 179, 180, 191, 258, 260, 283, 287, 294 schizophrenic, 255 secreted, 55, 90, 91, 105, 106, 111, 145, 146, 153, 165, 170, 182, 225, 227, 265, 285, 289, 292 self-cleaving, 146, 285 self-defeating, 259, 261, 290 sequestering, 9, 12, 15, 50, 68, 71, 72, 161, 167, 180 sheddases, 53, 258 size range, 257 stoichiometry, 13, 21, 61, 73, 163, 165, 171, 175, 286, 287, 291, 295, 298, 300, 303 sulphotransferases, 293 synthesis, 29 targeting, 20 tethering, 9, 12, 15, 24, 165, 286, 289 tetramers, 160 transcription factors, 18, 41, 48–52, 79, 105, 111, 132–136, 141, 143, 148, 156, 158, 163, 171, 180, 181, 186, 190, 213–224, 233, 239, 247–249, 254, 258–265, 272–293, 297, 300. See also under gene regulation transmembrane, 9, 15, 179, 180, 182, 191, 213, 259, 261, 272, 274, 275, 279–294 turnover rates, 126, 147, 286, 287, 295 two-hybrid assays, 21, 55 two-hybrid screens, xiii, 24, 160 ubiquitin ligase adaptors, 180, 260, 287, 294, 296 ubiquitin ligases, 182, 265, 272, 273, 282, 296 ubiquitous. See genes: ubiquitously expressed uncleavable constructs, 13, 287 weird, 21, 179, 213 proteins, common actin, 125, 174 cadherins, 90, 92, 153, 191, 258, 295, 304 clathrin, 26, 124, 146 collagen, 174 connectin, 149 cyclins, 29, 44, 49, 233, 258, 260, 280 dynamin, 26, 124 importins, 258 insulin, 92 integrins, 160, 173, 174, 177 kinesin, 11, 286 laminin, 174 myosin, 11

458

proteins, common (contd.) neurexins, 191 neuroglian, 149 spectrins, 180 tropomodulin, 11 ubiquitin, 180 proteins, modiﬁcation of, 21 by acetylation, 248, 279, 288, 295 by acylation, 285 by autophosphorylation, 290 by autoproteolysis, 285 by cholesterol adducts, 125, 146, 285 by cleavage, 9, 12–15, 53, 107, 111, 126, 146, 181, 182, 213, 217, 259, 265, 274, 285–289 by deacetylation, 17, 248, 249, 275, 276, 281 by dephosphorylation, 275, 294, 304 by geranylation, 180 by glycosylation, 15, 124, 146, 165, 261, 292 by hyperphosphorylation, 294 by phosphorylation, 9, 13, 15, 24, 107, 111, 126, 145, 180–183, 218, 235, 260, 263, 273, 286–296, 300, 303 by reshaping, 26, 258, 261, 264, 286, 290 by ubiquitination, 180, 182, 263, 280, 287, 294 proteins, particular. See genes, particular Rauskolb, Cordelia, 173 Ready, Donald, 202, 211, 212, 224 receptors afﬁnity for ligand, 52, 124, 125, 165, 293 auto-repression, 261 capping, 211 cassettes, 179 cis interactions, 164, 211 cleavage of precursor, 15, 213, 274 constitutively active, 12, 114, 124, 141, 145, 151, 170, 173, 190, 207, 216, 234 co-receptors, 105, 109, 217, 286, 290, 293 density, 125, 188 density, effects of, 147, 158 dimerization, 9, 181, 293 dominant-negative, 147, 169, 173, 183, 195, 216, 242 endocytosis, 27, 59, 124, 146, 182, 285 gradients, 147 heterodimers, 289 hypersensitized, 52, 189, 190 hyposensitized, 143, 189, 190 interchangeability, 216, 293 ligand alternation, 165 ligand-induced cleavage, 9, 12, 13, 15, 53 maturation in Golgi, 15, 165, 274 occupancy levels, 81 olfactory, 191 oligomerization, 9, 52, 261, 289 phosphorylation, 181 protein complexes, 179, 180, 273, 282, 286, 289, 293 proteolysis, 12, 13, 15, 124, 182, 286 recycling, 124 regulation by ligand, 125, 146, 158, 166, 288, 292, 295 regulation of ligand, 166 serine-threonine kinases, 290 trans-ingestion, 175 tyrosine kinases, 170, 179, 182, 213, 231, 259, 264, 290, 303

INDEX

unknown, 55, 134 with multiple ligands, 170 Reh, Thomas, 217 Reinitz, John, 79 Renfranz, Patricia, 225 Rhyu, Michelle, 5 RNA antisense, 303 binding, 264 clocks, 297 computation, 73 domains, 73, 272, 279 heteroduplexes, 73 localization, 73, 76, 84 maternal, 76 processing, 24, 73, 79 splicing, 18, 19 translation, 73 turnover, 73, 100 untranslated portion, 48, 73 Roegiers, Fabrice, 53 Romani, Susana, 43 Rubin, Gerald, 225, 252 rules, xi, xii, 270, 300. See also cell instructions about axon wiring, 55, 191–196, 199 about bracts, 29 about bristle patterns, 31, 68, 229, 230 about bristles, 20, 55 about cell autonomy, 47 about cell lineage, 1, 18 about cell size, 55, 56 about cellular automata, 236 about disc identity, 251, 252, 305 about distalization, 115, 119, 157, 158 about eye’s D-V axis, 203, 205, 209 about gene regulation, 18, 91, 104, 115, 171–173, 248, 251, 254 about growth vs. differentiation, 59, 118 about inhibitory ﬁelds, 55, 57, 59 about interfaces, 148, 154, 155 about ommatidia, 215, 227 about regeneration, 93, 94, 95, 96, 97, 100, 103, 156 about regulatory hierarchies, 136 about tissue continuity, 94, 254 about transdetermination, 84 about trespassing, 132 about Turing-like models, 69 about wing veins, 187 Bateson’s Rule, 99, 100, 101, 103, 153 Distalization Rule, 93 Dpp-Wg Intersection Rule, 157 Posterior Prevalence Rule, 248 Proximity-vs.-Pedigree Rule, 4, 5 Reciprocity Rule, 92, 94, 95, 96, 122, 156 Shortest Intercalation Rule, 93, 95, 101, 154, 156 Venn Overlap Rule, 172, 251 S´anchez-Herrero, Ernesto, 246 Schneiderman, Howard, xii, 32 Schubiger, Gerold, 122 Schultz, Jack, 38 science ad hoc assumptions, 99, 100, 103, 122, 131 artifacts, xiv, xv, 48, 57, 107, 144, 147, 279 big vs. little, 256 cleverness, 31, 51, 97, 100, 143, 224, 239, 246 conventional wisdom, 246

correlation without causation, 4, 119, 149, 157, 234. See also uncoupling counterintuitive facts, 35, 160, 163 debates. See history deductions, 12, 35, 37, 39, 43, 67, 83, 91, 124, 131, 170, 251, 298 delusions, semantic, 247 dialectic, 12, 187 disappointments, 33 discoveries, xi, 2, 4, 5, 35, 37, 40, 44, 49, 53, 85, 86, 90, 105, 121, 122, 125, 132, 149, 158, 165, 202, 208, 224, 256, 298 disproofs, 38, 39, 44, 48, 158, 185, 202, 211, 224, 229 dogmas, 53, 57, 96, 97, 246 dramatics, 21, 75, 85, 134, 135, 143, 165, 237 enigmas, 268–270 epiphanies, 270 heresies, 15, 41, 57, 96, 119, 149, 246, 285, 292 heuristics, 123, 136, 266, 302 hypotheses, 266–268 illusions, xv, 23, 68, 72, 134, 166 insights, xi, xv, 33, 56, 79, 83, 97, 139, 157, 246, 254, 256, 300 messy data, 43, 47, 183, 287 metaphors, 266–268 mistakes, xiv, 12, 38, 43, 147, 156 models, 266–268 mysteries, 268–270 myths, 147, 202 negative results, 147 paradigms, xv, 43, 44, 79, 83, 84, 103, 202, 245 paradoxes, 10, 27, 52, 55, 56, 80, 93–96, 113, 128, 160, 169, 183, 231, 246, 268–270, 282, 286 predictions, 10, 15, 33, 41, 44, 47, 49, 56, 79, 85, 97, 153, 205, 213, 218, 265 proofs, 1, 12, 20, 50, 61, 67, 86, 90, 113, 115, 118, 139, 155, 157, 202, 204, 212, 217, 231, 246, 297 prophecies, 43, 79, 84, 97, 190, 301 reasoning by analogy, 137, 148, 161, 163, 203 reasoning from ﬁrst principles, xii, 31, 35, 60, 101, 125, 158, 201, 213, 216 red herrings, 18, 109, 134 reductionism, 299, 302 riddles, 268–270 scenarios, 266–268 skepticism, 12, 100, 114, 187, 202 surprises, xii, 18, 33, 37, 41, 43, 44, 52, 68, 69, 77, 87, 91, 97, 99, 105, 131, 153, 180, 183, 209, 216–218, 243, 252, 260, 282, 287, 303–305 systems analysis. See uncoupling unnatural side-effects, 25, 124 self-assembly. See also boundaries, creation of; emergent properties; mechanisms; pattern formation; positional information general problem, xi of bristle rows, 65, 67, 299 of concentric rings, 134, 224, 304 of dynamin collars, 26 of multi-protein complexes, 248, 249, 303 of ommatidia, 212, 213, 215, 227 of stripes, 150, 151, 173, 174

INDEX

sensilla campaniform, 28, 63, 65, 99, 131, 185, 276, 299, 304 chordotonal, 27, 29, 74, 112 external, 27 extra, 73 haltere, 246 identity genes, 27, 75 larval, 9, 24, 27 nests on legs, 304 olfactory, 27–29, 75, 191, 199, 246, 276 role of AS-C, 44 shifts, 63, 131 stretch receptors, 28 trichoid, 28, 304 vs. bristles, 27–28 vs. bristles on wing vein L3, 187 wing, 174 wing radius, 18 sensory organ precursor (SOP). See also bristles ablation, 49 asynchrony, 55, 59, 62 computer, 72 delamination, 65 eccentric, 43, 47 ﬁlopodia, 53, 65, 67, 299 heterochronic pairs, 53, 57 initiation, 21, 37, 43–55, 59, 61, 68, 71–73, 163, 229, 278–284 markers, 156, 282, 284 mitoses, 5, 7, 41, 49, 59, 92, 229 movements, 61, 63, 65, 67 patterns, 39, 59, 61, 94, 139, 163, 193 pre-SOP state, 47, 53 regression, 53, 71 repulsion, 65, 67 selection, 45–74, 229, 304 sequence, 41, 57, 59, 67, 133 vs. PNC identity, 48, 51 vs. R8p photoreceptor, 225 Serebrovsky, A. S., 37 Serrano, Nuria, 124 Shearn, Allen, xii signaling pathways. See also signaling pathways, particular antagonistic, 74, 114–117, 121, 167, 179, 234, 235 anti-mitogenic, 92 artiﬁcial switching, 171 as networks, 179, 300, 303 assays for activity of, 126, 183, 218 branched, 181, 293 comparisons, xii, 27, 92, 105, 109, 157, 179–182, 297, 300, 303 context-dependence, 21, 179, 181, 303 control of AS-C, 71 cross-talk, 15, 50, 52, 129, 136, 167, 180, 274, 279, 284, 290, 294, 300, 303 mitogenic, 92 PCP (planar cell polarity), 131 shared components, 15, 109, 157, 180, 181, 275 unorthodox variations, 11, 109, 173, 289, 292 signaling pathways, particular Boss-Sevenless, 179, 181, 213, 215, 218, 220, 233, 303 dActivin, 92

459

Decapentaplegic, 105, 109, 124, 132, 141, 157, 167, 173, 177–180, 185–190, 194, 231, 235, 286, 289–292 EGFR, 29, 48, 52, 74, 92, 107, 153, 169, 170, 174, 177–189, 194, 216–219, 227–229, 233, 235, 242 Hedgehog, 92, 105, 109–113, 149, 154, 157, 177, 185–187, 194, 208, 231, 235, 242, 285–289 insulin receptor, 92 JAK-STAT, 92, 205, 303 JNK, 118, 263 Notch, 9–27, 49–55, 68, 69, 73–75, 92, 135, 159–169, 173–180, 188, 194, 203–211, 218, 219, 223–229, 235, 242, 263, 272–275, 279–284, 303 PCP (planar cell polarity), 293 Ras-MAPK, 92, 179, 216, 218, 224, 263, 304 RTK, xiv, 170, 179–182, 218, 231, 258, 264, 265, 303 Wingless, 15, 52, 92, 105, 109, 132, 136, 156, 157, 167, 169, 181, 182, 189–190, 194, 205, 242, 245, 279, 285–288, 292–296 Simpson, Pat, 52 Skeath, James, 41 Spemann, Hans, 36 Spencer, Susan, 227 Stern, Curt, xv, 32–43, 49, 56, 57, 75, 79–83, 86, 163, 164, 190, 246, 251, 252, 256, 298–302 Strigini, Maura, 137 Struhl, Gary, 157, 211, 224, 231, 234 Strutt, David, 231 Sturtevant, Alfred Henry, 1, 2, 4, 31, 38, 39, 86, 91, 134, 298, 299 Swammerdam, Jan, 76 Thomas, John, 173 Tokunaga, Chiyoko, 80, 83, 252 Tomlinson, Andrew, 211, 212, 224 topology disc-exoskeleton, 89, 99, 139, 199 embryo-larva, 77, 89 eye-wing, 203 gene-anatomy, xi, 20, 31, 41, 45, 239, 242, 297, 298, 301 gene-cell type, 83 larva-adult, 2 leg disc vs. body segment, 89, 109, 114 leg segment vs. body segment, 136 leg-antenna, 83, 199, 251, 299 leg-wing, 137 neuro-sensory, 5, 191 space-time, 41, 43 wound healing, 96 transdetermination, 84–86, 96, 123, 169, 242, 249, 254, 302 Turing, Alan, 33 typeface formats, xiii, xiv uncoupling. See also circuitry of border from P compartment, 125, 151 of cell size from body size, 55, 56 of chemical reactions, 73 of early vs. late roles of Dpp in wing, 188 of growth from differentiation, 196 of growth from patterning, 141, 158 of growth in adjacent compartments, 92, 301 of identity from afﬁnity, 173

of identity from boundaries, 154 of identity from signaling, 149, 161 of individuation from segmentation, 84, 299 of larval from adult development, 86 of ligand binding from signaling, 285 of ligand transport from signaling, 125 of optic stalk from eye furrow, 234 of pedigrees from patterning, 3, 4 of sensilla from veins, 185, 186 of signaling from polarity, 205 of veins from crossveins, 185, 189 of veins from one another, 185, 186 Ursprung, Heinrich, xii Waddington, C. H., 32, 224, 299, 300, 304 Weatherbee, Scott, 245 Weinstein, Alexander, 37 Weismann, August, 76 Weiss, Paul, 299, 302 White, Richard, 212 Whiting, Anna, 302 Wieschaus, Eric, 76 Wigglesworth, Vincent, 32, 49, 57, 59, 224 Wilkins, Adam, 97 wing airfoil, 136, 167, 174 alula, 186 anatomy, 139, 159, 177 axes. See under axes blade, 49, 55, 87, 105, 153, 157, 173, 174, 185, 189, 190 blade vs. hinge identity, 139, 158, 172, 301, 302 butterﬂy, 31, 299 corrugation, 174 duplications, 123, 157 evolution, 86 expansion, 174 extra, 141 geometry, 137, 189 growth, 29 hairs, 87, 159, 189, 205 hinge, 132, 139, 141, 172, 190, 240, 296, 301 hinge, overgrowth of, 158 identity (“wingness”), 171, 240, 249, 254 margin. See wing margin missing, 137, 140, 151, 159, 160, 167, 303 moth, 61, 65, 132 notches, 157, 164, 165 sensilla, 185 shape, 164 size, 29 small, 140, 147, 151–154, 164, 185 symmetry, A/P, 188 transalar adhesivity, 174 transalar lumen, 174 veins. See wing veins wing disc advantages (vs. other discs), 93, 105 bipolar duality, 114, 167–173, 302 compartments, 4, 87 distalization, 157, 171 duplications, 157 evagination, 174 extra, 170 fate map, 94, 139, 156, 193, 301 folds, 87, 126, 139 gene expression patterns, 89, 111, 141, 142, 169, 173, 177, 193, 245

460

wing disc (contd.) geometry, 137, 139, 160, 301 growth, 29 initiation, 77, 89, 91, 171 margin. See wing margin margin syndrome, 164, 165 mitoses, region-speciﬁc, 92 nomenclature, xiii outgrowths, 148, 153, 155, 157 outgrowths, multiple, 157 spot, unique distal, 157 spot, vg-on (in notum), 158 veins. See wing veins vs. leg disc, 137, 170, 171 wing vs. notum portion, 87, 167–173, 190–191, 193 wing margin asymmetry of, 159, 160, 164 at the eye equator (sic! ), 203 bracts, 28 bristle rows, 31, 65, 67, 159, 189 bristles, 27, 48, 139, 159, 167, 277, 279, 282, 302 cell afﬁnities in, 173 cell types in, 174 creation of, 159, 160, 173, 302 ectopic, 96, 159–161, 164, 165 gene expression in, 27, 48, 141, 146, 159, 163–167, 177, 183, 301, 303 gene regulation in, 161, 164, 171, 179, 295, 296 geometry, 137, 159 growth control by, 153, 157 missing, 284, 302 mitotic quiescence, 92 mutations affecting, 160 nomenclature, 153, 154, 155 tip vs. remainder, 157 vein properties of, 139, 183, 189

INDEX

vs. blade, 141, 173 vs. haltere capitellum, 245 wing pouch asymmetry vs. symmetry, 167, 301 circuitry, 163, 166 dorsal edge, 128 extra, 157, 158, 170 gene expression in, 92, 141, 159, 171, 174, 177, 301 gene regulation in, 129, 148, 173, 177, 245, 288, 292, 296, 301 gradients, 142, 146 growth, 164 homeosis, 240 initiation, 87, 167, 170, 171 morphogens, 140, 142, 146, 156 overgrowth, 157, 164, 165 response to Dpp, 147 response to Notch, 164, 167 size, 139, 142 wing veins annealing, 137, 174, 175, 177, 245 asymmetry, 174 atavistic, 174 branching, 173 circuitry (vs. tracheae), 174 conﬂuent lawns, 183, 185, 186, 188 corrugations, 160 cross-veins, 177, 185, 188–189 determination, 188 displacements, 185, 186 extra, 173, 174, 182–188, 303 formula, 139, 154 function, 174 fusion, 153, 173, 185 gene expression in, 177, 183, 186–189 gene regulation in, 177 inducers vs. suppressors, 183, 186, 187 initiation, 182, 183

initiation vs. ﬁne-tuning, 177 interveins, widening of, 185 irregular, 188 L1. See wing margin L2 and L5 (Dpp-dependent), 177, 185–188 L2 sprouting bristles, 187 L3 and L4 (Hh-dependent), 177, 185–186 L3 proneural stripe and sensilla, 185 lateral inhibition, 175, 188 markers, 189 missing, 182, 183, 185–188 mitotic zones, 175 nomenclature, 177, 189 pattern in disc vs. adult wing, 94, 139, 193 patterning, 153–156, 173–191 pigmentation, 174 proveins, 175, 177 regulation by Delta-Notch, 53, 175–177, 188 regulation by Dpp, 177, 185, 188 regulation by EGFR, 177–185, 188 sharpening, 184 straightening, 173, 177 vein vs. intervein identities. See under cell states vs. bristles, patterning, 175, 187 vs. interveins, 174 vs. mesectoderm stripes, 179 vs. paraveins, 174 widening, 175, 182, 188 widening at tips, 12, 175 widths, 167, 173, 177 Wolff, Tanya, 211 Wolpert, Lewis, 32, 79, 80, 81, 83, 84, 93, 248, 249, 251, 299 Zecca, Myriam, 137, 157 Zimm, Georgianna, xii

Imaginal Discs: The Genetic and Cellular Logic of Pattern Formation (Developmental and Cell Biology Series)

Morphogenesis: The Cellular and Molecular Processes of Developmental Anatomy (Developmental and Cell Biology Series)

Morphogenesis: The Cellular and Molecular Processes of Developmental Anatomy (Developmental and Cell Biology Series)

Meiosis (Developmental and Cell Biology Series)

Understanding Ageing (Developmental and Cell Biology Series)

Fungal Morphogenesis (Developmental and Cell Biology Series)

Dictyostelium: Evolution, Cell Biology, and the Development of Multicellularity (Developmental and Cell Biology Series)

Cellular Automaton Modeling of Biological Pattern Formation

Cytokinesis in Animal Cells (Developmental and Cell Biology Series)

The Zebrafish: Cellular and Developmental Biology, Part B, Volume 101, Third Edition (Methods in Cell Biology)

The Zebrafish: Cellular and Developmental Biology, Part A, Volume 100, Third Edition (Methods in Cell Biology)

The Consequences of Chromosome Imbalance: Principles, Mechanisms, and Models (Developmental and Cell Biology Series)

The formation and logic of quantum mechanics

Formation and Logic of Quantum Mechanics: Formation of Atomic Models

Receptors of Cell Adhesion and Cellular Recognition

Board Review Series: Cell Biology and Histology

Histology and Cell Biology

Board Review Series: Cell Biology and Histology

Models of Biological Pattern Formation

Cellulose. Molecular and Cellular Biology

Brucella: Molecular and Cellular Biology

This Side Up: Spatial Determination in the Early Development of Animals (Developmental and Cell Biology Series)

This Side Up: Spatial Determination in the Early Development of Animals (Developmental and Cell Biology Series)

Endosperm: Developmental and Molecular Biology (Plant Cell Monographs, Volume 8)

Plant Cell Monographs Endosperm. Developmental and Molecular Biology

Yersinia: Molecular and Cellular Biology

Cellular Genetic Algorithms

Cellular genetic algorithms

The Dictionary of Cell and Molecular Biology

Pattern Formation at Interfaces

Developmental Biology

Imaginal Discs: The Genetic and Cellular Logic of Pattern Formation (Developmental and Cell Biology Series)

Morphogenesis: The Cellular and Molecular Processes of Developmental Anatomy (Developmental and Cell Biology Series)

Morphogenesis: The Cellular and Molecular Processes of Developmental Anatomy (Developmental and Cell Biology Series)

Meiosis (Developmental and Cell Biology Series)

Understanding Ageing (Developmental and Cell Biology Series)

Fungal Morphogenesis (Developmental and Cell Biology Series)

Dictyostelium: Evolution, Cell Biology, and the Development of Multicellularity (Developmental and Cell Biology Series)

Cellular Automaton Modeling of Biological Pattern Formation

Cytokinesis in Animal Cells (Developmental and Cell Biology Series)

The Zebrafish: Cellular and Developmental Biology, Part B, Volume 101, Third Edition (Methods in Cell Biology)

The Zebrafish: Cellular and Developmental Biology, Part A, Volume 100, Third Edition (Methods in Cell Biology)

The Consequences of Chromosome Imbalance: Principles, Mechanisms, and Models (Developmental and Cell Biology Series)

The formation and logic of quantum mechanics

Formation and Logic of Quantum Mechanics: Formation of Atomic Models

Receptors of Cell Adhesion and Cellular Recognition

Board Review Series: Cell Biology and Histology

Histology and Cell Biology

Board Review Series: Cell Biology and Histology

Models of Biological Pattern Formation

Cellulose. Molecular and Cellular Biology

Brucella: Molecular and Cellular Biology

This Side Up: Spatial Determination in the Early Development of Animals (Developmental and Cell Biology Series)

This Side Up: Spatial Determination in the Early Development of Animals (Developmental and Cell Biology Series)

Endosperm: Developmental and Molecular Biology (Plant Cell Monographs, Volume 8)

Plant Cell Monographs Endosperm. Developmental and Molecular Biology

Yersinia: Molecular and Cellular Biology

Cellular Genetic Algorithms

Cellular genetic algorithms

The Dictionary of Cell and Molecular Biology

Pattern Formation at Interfaces

Developmental Biology

Recommend Documents